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Formation of neuromuscular junctions in adult rats: Accumulation of acetylcholine receptors, acetylcholinesterase, and components of synaptic basal lamina
DEVELOPMENTAL
BIOLOGY
84,255-266
(1981)
Formation of Neuromuscular Junctions in Adult Rats: Accumulation of Acetylcholine Receptors, Acetylcholinesterase, and Components of Synaptic Basal Lamina CRISPIN B. WEINBERG, Neurobiology
JOSHUA R. SANES,’ AND ZACH W. HALL
Division, Department of Physiology, University of Califomzia School of Medicine, San Francisco, California Department of Neurobiology, Harvard Medical School, 25 Shattuck St., Boston, Massachusetts 02115
9/l&?,
and
Received August 21, 1980; accepted in revised form November 17, 1980 We describe the appearance and accumulation of four specialized molecular components of the adult neuromuscular junction during ectopic endplate formation in adult rat soleus muscles. One component, the acetylcholine receptor (AChR), is a major constituent of the postsynaptic muscle membrane. The other three, acetylcholinesterase (AChE) and two extracellular synapse-specific antigens, are associated, at least in part, with the basal lamina in the synaptic cleft. The accumulation of each component was studied by immunocytochemistry. In addition, the accumulation of AChRs was measured by autoradiography after reaction with ‘%I-a-bungarotoxin, and AChE was examined by histoehemistry and by sedimentation analysis. Endplate formation was initiated by cutting the original nerve to a muscle in which a foreign nerve had been previously implanted. Within 2 days, clusters of AChRs appeared in the new endplate zone. The density of AChRs in these clusters increased from 1 X lo4 sites/pm’ to nearly the final value of 2 X 10’ sites/ pm2 by 4 days. The cluster continued to grow in size and receptor number over the next month. AChE was not detected on the surface of the muscle fiber until after 1 week, when it was present at some, but not all ectopic endplates. Its appearance coincided with a rapid accumulation of the endplate-specific, 16 S form of AChE (A& in the portion of the muscle containing new endplates. By 2 weeks virtually all endplates stained for AChE and by 1 month both immunochemical and histochemical staining resembled that of normal adult endplates. The synapse-specific basal lamina antigens that we studied were detected at some endplates by Day 6, and their further appearance followed a time course similar to, or slightly ahead of, that of AChE. Thus maturation of the synaptic basal lamina occurs after the AChRs have formed clusters and achieved nearly their final density.
INTRODUCTION
are assembled and because the synaptic basal lamina has been shown to influence the differentiation of preand postsynaptic membranes during regeneration of damaged nerve and muscle fibers (Sanes et al., 1978; Burden et al., 1979). We wished to examine the temporal relation between the assembly and macromolecular differentiation of the synaptic basal lamina and of the postsynaptic muscle membrane during the formation of the NMJ. We chose to study the ectopic synapses that are formed between transplanted “foreign” motor axons and denervated soleus muscle fibers in the adult rat. During spontaneous reinnervation, motor axons preferentially reinnervate muscle fibers at original synaptic sites (Gutmann and Young, 1944; for review see Bennett and Pettigrew, 1975). In contrast, when a nerve is implanted far from the original endplates, and the original innervation is interrupted, new endplates are formed at ectopic sites on previously nonsynaptic portions of the muscle fiber surface (Elsberg, 1917; Aitken, 1950; Guth and Zalewski, 1963; Miledi, 1963; Fex et al., 1966; Jansen et al., 1973). In this case, specializations of the postsynaptic muscle membrane and of the synaptic cleft are formed de novo. The physiological (Jan-
At the adult neuromuscular junction (NMJ), both the postsynaptic muscle membrane and the extracellular basal lamina in the synaptic cleft are biochemically specialized. Acetylcholine receptors (AChRs) are clustered in the postsynaptic membrane, and acetylcholinesterase (AChE) is highly concentrated in the synaptic cleft where at least some of it is associated with the basal lamina (Hall and Kelly, 1971; Betz and Sakmann, 1973; McMahan et al., 1978). In rat muscle, a particular form of AChE (16 S) appears to be specifically associated with endplates (Hall, 1973; Vigny et al., 1976). Several other components of the basal lamina have been shown by immunological methods also to occur at high density in the synaptic cleft, but not elsewhere on the muscle fiber’s surface (Sanes and Hall, 1979). The relation between the differentiation of the basal lamina and of the postsynaptic membrane during synapse formation is of interest both in terms of understanding how specialized components of the cell surface ’ Present address: Department of Physiology and Biophysics, ington University School of Medicine, St. Louis, MO. 63110
Wash-
255 0012-1606/81/080255-12$02.00/O Copyright All rights
0 1981 by Academic Press, Inc. of reproduction in any form reserved.
256
DEVELOPMENTAL
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sen et al., 1973; Frank et al., 1975; LQmo and Slater, 1976,19’78) and morphological (Korneliussen and Sommerschild, 1976; Waerhaug et al., 1977) events that occur during the formation of ectopic NMJs in the rat soleus muscle have been well described and form a useful framework in which to investigate molecular aspects of synaptogenesis. We describe here the accumulation and differentiation of AChR, AChE, and two synaptic basal lamina antigens during the formation of ectopic NMJs in adult rats. Our principal finding is that the differentiation of the postsynaptic muscle membrane and of the synaptic basal lamina do not occur together but follow different time courses. METHODS
Ectopic Endplates. Ectopic innervation of rat soleus muscles was produced as described by Frank et al. (1975). Sprague-Dawley rats (100-200 g) were anesthetized with sodium pentobarbital and the superficial fibular nerve was dissected free in the lower leg and cut at the ankle. The cut end of the nerve was placed on the proximal portion of the soleus muscle, at least 5 mm away from the original endplates, which occupy a band in the middle of the muscle. The overlying muscle was then sutured and the incision was closed with wound clips. To initiate synaptogenesis, the soleus muscle was denervated by removing a 5- to lo-mm segment of the tibia1 nerve 2 to 4 weeks later (= Day 0). At appropriate intervals thereafter, animals were killed and their soleus muscles removed for histochemical or biochemical analysis. In some cases, to monitor the progress of ectopic innervation, the muscles were dissected in oxygenated Kreb’s solution, the foreign nerve was stimulated with a suction electrode, and tension was measured with a Grass force transducer. Ectopic innervation failed to develop in about 10% of the animals. Histology. Nerve fibers and sites of AChE activity were examined in lOO-pm longitudinal cryostat sections of the entire soleus muscle. The sections were stained by combined cholinesterase histochemistry and silver impregnation of nerve fibers (Beerman and Cassens, 1976). Immunocytochemistry. The accumulation of synapsespecific molecules was studied immunocytochemically, using antisera to rat skeletal muscle AChR (Weinberg and Hall, 1979b), bovine brain AChE (Greenberg et al., 1977; kindly provided by Drs. A. Greenberg and A. Trevor of UCSF), bovine lens capsule (Sanes and Hall, 1979), and a basement membrane collagen-rich fraction from rat muscle (Sanes and Hall, 1979). The antibasement membrane collagen was adsorbed before use with
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connective tissue from endplate-free regions of rat diaphragm, in order to reveal synapse-specific antibodies (Sanes and Hall, 1979); the other sera was not adsorbed before use. Anti-AChR was used at a dilution of 1:500 in phosphate-buffered saline (PBS) containing 10 mg/ ml BSA, while other sera were diluted 1:200. Immunocytochemical methods have been detailed by Sanes and Hall (1979). Briefly, cross-sections of frozen, unfixed muscle were cut in a cryostat, incubated first with antiserum and then with a mixture of fluorescein-conjugated goat anti-rabbit IgG (GAR, Cappel) and rhodamine-a-bungarotoxin (rhodamine-BuTx, a gift from Drs. Peter Ravdin and Darwin Berg of University of California, San Diego), and finally viewed through selective filters that allowed us to see fluorescein and rhodamine separately. The rhodamine-BuTx permitted us to verify the identity of antibody-stained endplates, and also provided an additional way of assessing the distribution of AChRs at ectopic synapses. Although immunocytochemical studies of developing synapses do not provide a quantitative measure of antigen concentrations, we desired to know if the time at which we first detected synapse-specific antigens was determined entirely by the sensitivity of our method (as would occur if the antigen concentration rose gradually until it finally reached the threshold for detection) or whether the antigen was present at a concentration well above the sensitivity of our method on the first day we could detect it (in which case the “appearance” of an antigen would represent a more or less abrupt rise in its concentration). To distinguish between these two possibilities, we performed an experiment using, as second antibody, mixtures of fluorescein-conjugated and unconjugated GAR in varying proportions with the total titer of antibody kept constant. The nonspecific background staining is reduced along with the specific staining, as the concentration of fluorescein is decreased. Thus the precise value of the dilution of fluorescein-GAR that gives just-visible staining is not significant; however, this test can show whether antigens are present in concentrations that are well above the limit of detection. At adult endplates, binding of antibodies to antigens in the synaptic basal lamina was easily detected when a 1:8 mixture of fluorescein-conjugated GAR and unconjugated GAR was substituted for the usual amount of fluorescein GAR. Autoradiography. AChRs were quantitated by autoradiography of single muscle fibers labeled with ‘%ICX-BuTx as described previously (Reiness and Weinberg, 1981). A nearly saturating dose of 1251-~-B~T~ (lo-12 cLg/lOOg body wt) was injected into the femoral artery of an anesthetized rat. Three hours after labeling, the soleus muscle was removed, fixed, and reduced to single fibers in a VirTis homogenizer. The fibers were dried
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ET AL.
Adult Rat Neuromuscular
onto gelatin-coated slides which were then dipped in Kodak NTB-2 emulsion, exposed at 4”C, developed, and examined under dark-field illumination. Silver grains in each cluster were counted and the area of the cluster was measured. The number of toxin binding sites was calculated assuming that 20% of the ‘=I emissions produced a grain (Burden, 1977). For normal soleus endplates we obtained values of 1.4 +- 0.2 X 10’ receptors per endplate and a density of 2.7 ? 0.2 X lo* receptors/ pm2 (mean f SEM, n = 6) which are in good agreement with values obtained in other rat skeletal muscles (reviewed in Edwards, 1979). In one control experiment we visualized the same endplates by both autoradiography and immunocytochemistry. Muscles that had been labeled with ‘25I-aBuTx in vivo were dissected, fixed lightly (4% formaldehyde for 10 min at room temperature), reduced to single fibers, and stained with anti-AChR. After endplates were located and photographed, the muscle fibers were subjected to autoradiography. Five endplates were later relocated and photographed under dark-field illumination. The images obtained by the two techniques were superimposable and the regions of most intense fluorescence generally corresponded to regions of highest grain density (Fig. 1). Sedimentation analysis of acetylcholinesterase. The forms of AChE were separated by sedimentation on sucrose gradients as described previously (Hall, 1973; Weinberg and Hall, 1979a). The ectopically innervated region of the soleus was separated from the region containing the original endplates, homogenized in ice-cold buffer (1 M NaCl, 1% Triton-X-100, 50 mM Tris-HCl, and 0.2 mM EDTA, pH 7.4), and centrifuged at 15,000g for 15 min at 4°C. Aliquots of the supernatant were run on linear sucrose gradients (5-20s). AChE activity was assayed by the method of Ellman et al. (1961) and under
FIG. 1. Correspondence of immunocytochemistry raphy. The same endplate on a single muscle immunocytochemical staining with anti-AChR (B). Bar = 20 Wm. radiography of ‘251-~-B~T~
and autoradiogfiber is shown after (A) and after auto-
Junctions
257
the conditions used was directly proportional to the enzyme concentration. Control experiments with inhibitors of specific and nonspecific cholinesterases, (BW234c51) and tetramonoisopropyl pyrophosphortetramide (iso-OMPA), respectively, showed that nonspecific cholinesterases accounted for less than 15% of the activity in each peak on gradients of these extracts. RESULTS
Development
of Ectopic
Endplates
Ectopic NMJs were produced on denervated soleus muscle fibers by axons of a foreign nerve, the fibular nerve. In a first operation, the fibular nerve was implanted on an endplate-free zone of the soleus. Two to four weeks later, ectopic synapse formation was initiated by cutting the original nerve (= Day 0). Ectopic synapses are more numerous and develop more quickly when this schedule is used than when a foreign nerve is implanted on a muscle at the same time that the original nerve is cut (Fex and Thesleff, 1967). Neither structural nor functional signs of ectopic synapse formation were seen in muscles before the original nerve was cut (see also Elsberg, 1917; Aitken, 1950; Guth and Zalewski, 1963). In silver-stained sections of normally innervated muscles with an implanted foreign nerve, foreign nerve fibers were seen growing along muscle fibers, but these axons rarely branced and had few growth cones (see also Aitken, 1950). No contractions were recorded from such muscles when the foreign nerve was stimulated, and accumulations of AChRs or AChE were generally not seen on soleus muscle fibers in the region of the fibular nerve implant. One control muscle did contain a few endplates on fibers that lay directly under the foreign nerve; these synapses were presumably formed on muscle fibers that had been injured during the implantation operation and thus rendered susceptible to hyperinnervation (Miledi, 1963, Gwyn and Aitken, 1964). Ectopic synapses formed quickly once the original nerve was cut. Three days after denervation, implanted muscles had larger numbers of highly branched foreign nerve fibers with many growth cones (see, e.g., Fig. 5A). Korneliussen and Sommerschild (1976) have described vesicle-laden terminal boutons closely apposed to muscle fibers at 3-5 days. We first saw contraction of a few muscle fibers in response to stimulation of the foreign nerve on Day 3. This is consistent with the observation that spontaneous and evoked endplate potentials are first seen 2 l/2 to 3 days after denervation (Lomo and Slater, 1976,1978; see also Fex and Thesleff, 1967). Thus morphological and physiological evidence of ectopic synapse formation is present within 3 days of denervation.
258
DEVELOPMENTAL
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The average strength of contractions evoked by stimulating the foreign nerve increased gradually over the next 2-3 months (see also Fex and Thesleff, 1967; Frank et al., 1975). Foreign nerve terminals visualized by silver impregnation arborized and developed more extensive contacts with muscle fibers during this period. Subsynaptic folds have been reported at 10 days and become more extensive over the next 2 months (Korneliussen and Sommerschild, 1976). Ectopic endplates resemble normal adult endplates both morphologically and physiologically by 2-3 months. Acetylcholine
Receptors
When cross-sections of normal soleus muscle were stained with anti-AChR and fluorescein-GAR or with rhodamine-BuTx, the endplates appeared as bright broad bands or groups of spots (Sanes and Hall, 1979). The patterns of staining observed with rhodamineBuTx and with antiserum to AChR were identical, while muscles treated with preimmune sera and fluoresceinGAR showed little fluorescence above background. Only AChRs clustered at high density were revealed by these methods; extrajunctional receptors in embryonic or denervated adult muscles were not seen. In ectopically innervated soleus muscles, AChR clusters were first seen at 2 days. They appeared as faint, poorly defined patches of fluorescence on the muscle fiber surface that were coincident when seen under fluorescein and rhodamine optics (Figs. 2A, B), and were found only in the region of the soleus underlying the
FIG. 2. Accumulation of AChRs at ectopic endplates shown were stained with anti-AChR followed by fluorescein-second fluorescein optics. (B) is the same field as (A) photographed 6 days (D), and 16 days (E) after cutting the original nerve.
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84, 1981
foreign nerve. These presumably correspond to the patches of high ACh sensitivity found under the foreign nerve at 2 l/2 to 3 days (LQmo and Slater, 1976,1978). Because the staining of newly formed clusters was faint, the combined antibody and toxin labeling gave much greater sensitivity than either method alone; spots stained lightly, and presumably nonspecifically, by only one reagent or the other were found throughout the muscle. The AChR clusters seen with fluorescent antibodies and rhodamine-BuTx were clearly different from the “hot spots” that appear in extrajunctional regions of muscles several days after denervation (Ko et al., 1977). In rat soleus muscles the hot spots are typically 1 Mm2 and are almost never larger than 20 lrn2 (Ko et al., 1977), while by 2-3 days, the clusters that we identify as ectopic endplates have areas of 160-210 pm2. Hot spots were seen in autoradiographs of ectopically innervated muscles but were much rarer than in control (nonimplanted) denervated muscles. In ectopically innervated muscles, they were almost never seen more than 10 days after cutting the original nerve; this suggests that they were suppressed, as are unclustered extrajunctional AChRs (L6mo and Slater, 1976, 1978), by innervation. Three to four days after denervation patches of AChRs were more frequently seen and appeared in cross-section as short thin lines (Fig. 2C). By 1 week the lines had become longer but remained thin (Fig. 2D). During the second week the ectopic clusters became brighter and thicker so that by 16 days they were sim-
by immunofluorescence. Cross-sections of ectopically innervated soleus muscles antibody and rhodamine-bungarotoxin. (A) and (c-F) were photographed with with rhodamine optics. Eetopie endplates are shown 2 days (A, B), 3 days (C), (Ff shows normal endplates. Bar = 20 pm.
WEINBERG
ET AL.
Adult Rat Neuromuscular
ilar in appearance to, but smaller than mature synapses. At 1 month (Fig. 2E) they were indistinguishable from normal endplates. The thickening of the lines of staining may correspond to the formation of subsynaptic folds which starts during the second and third weeks (Korneliussen and Sommerschild, 1976). Autoradiography of muscles labeled with lWI-cr-BuTx confirmed the results obtained with anti-AChR and rhodamine-BuTx, and enabled us to determine the number and density of AChRs at developing ectopic endplates. Autoradiographs of some ectopic endplates are shown in Fig. 3 and the data obtained from these experiments are summarized in Table 1. Small, low-density clusters were first seen as early as 2 days. The average number of AChRs in a cluster steadily increased over the next month to achieve a value close to that found in adult endplates. The AChR density nearly doubled between the second and fourth days, and thereafter increased only slightly (Table 1, Fig. 4A). Thus the total number of AChRs in each cluster increases largely through the expansion of the total area occupied by the cluster. In experiments reported in the accompanying paper, we have found that the rate at which clustered AChRs are degraded changes between 2 and 6 days after denervation from an initially rapid value to a slower one characteristic of AChRs at adult endplates (Reiness and Weinberg, 1981). The time course of this change is roughly similar to that of the AChR density and, for comparison, is shown in Fig. 4B. Thus two of the important characteristics of AChRs at adult endplates, their high density and their slow rate of metabolic turnover, are well established at ectopic endplates within a few days after clusters of AChRs first appear. Many of the muscle fiber segments (typically l-mm long) observed during the first week had more than one
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259
cluster of AChRs. The number of fibers with more than one cluster decreased over the next few weeks until, by 3 weeks, most fibers had only one receptor cluster. This decrease presumably corresponds to the decrease in multiple innervation at newly formed ectopic NMJs that has been observed using morphological and electrophysiological techniques (Aitken, 1950; Frank et al., 1975; Kuffler et al., 1977). In summary, AChRs accumulate at ectopic NMJs at the same time as, or slightly before, the onset of synaptic transmission. Within a few days the clustered receptors are as metabolically stable and as densely packed as AChRs at mature NMJs. The size of the clusters, however, continues to increase for several weeks.
Acetylcholinesterase Although AChE is present throughout muscle fibers, its concentration at the mature NMJ is at least lOOOfold greater than in extrasynaptic regions (Hall, 1973). In addition, in rat muscle, a particular form of the enzyme, distinguished by velocity sedimentation (the 16S form) is specificially associated with the endplate region (Hall, 1973; Vigny et al., 1976). Both histochemical staining for AChE (see also Guth and Zalewski, 1963; Gutman and Hanzlikova, 1967) and the endplate form of the enzyme (Vigny et al., 1976) have been described at mature ectopic synapses. We therefore examined the accumulation of AChE and its molecular forms during the development of ectopic synapses. No AChE reaction porduct could be seen associated with the foreign nerve in normally innervated soleus muscles (Guth and Zalewski, 1963) or during the first 4 days after cutting the original nerve (Fig. 5A). The first change in the staining pattern of ectopically in-
FIG. 3. Accumulation of AChRs at ectopic endplates shown by autoradiography. Muscles which had been labeled with ‘%I-a-BuTx reduced to single fibers and processed for autoradiography. Dark-field photomicrographs are shown of eetopic endplates 2 days (A), (B), and 40 days (C) after cutting the original nerve, and of a normal endplate (D). Bar = 20 pm.
were 8 days
260
DEVELOPMENTAL
TABLE ACETYLCHOLINE Days after denervation
Note.
Ectopic
endplates
2 3 4 6 12 17 40
Normal
endplates
0
Means
k SEM
(n = 3-8; usually
RECEPTORS
1.7 3.2 4.4 5.7 8.9 12.3 13.2
10
Days
20
* f f + f f k
34, 1981
1 AT ECTOPIC
0.1 0.4 0.2 0.2 0.4 1.0 1.5
x X x x x X X
ENDPLATES Density (AChRs/rm’)
Number of AChRs/cluster lo6 lo6 lo6 lo6 lo6 lo6 lo6
14.0 + 1.5 x lo6
1.1 1.5 1.7 1.5 1.9 2.2 2.0
k f + f + * f
0.1 0.2 0.3 0.2 0.1 0.2 0.2
Area (rm’) x x x x x x x
lo4 lo4 lo4 10’ lo4 10’ lo4
2.7 k 0.2 X lo4
160 210 260 380 470 560 660
+ + k f + k ct
15 20 30 60 30 40 50
520 ck 50
4-6).
nervated muscles was observed on Day 5 when the region of the muscle fiber underneath some nerve endings and growth cones was diffusely stained (Fig. 5B). This staining appeared to be cytoplasmic and may represent the accumulation of AChE in subsynaptic cisternae prior to its deposition on the muscle fiber surface (Lentz, 1969; Wake, 1976). The first focal staining for AChE under nerve endings was observed 1 week after denervation (Fig. 5C, Lbmo and Slater, 1976, 1978). Stained areas appeared as the single “ovoid cups” described by Koenig (1971). Two weeks after denervation the patches were darker and more sharply defined (Fig. 5D), and by l-2 months they formed groups of spots or ovoid cups (Fig. 5E) which resembled normal endplates (Fig. 5F). The increase in number of patches or ovoid cups at single ectopic endplate probably corresponds to the increase in the number of synaptic boutons described by Korneliussen and Sommerschild (1976) during the second and third weeks after cutting the original nerve.
0
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30
40
Normal
after denewation
FIG. 4. Density and turnover of AChRs at ectopic endplates. Ectopically innervated muscles were labeled with ‘Z51-a-BuTx in V&O and processed for autoradiography at various times after labeling. The density (A) and mean half-time for turnover (B) for clustered receptors are plotted as a function of the time after cutting the original nerve at which the muscle was labeled. (Turnover times from Reiness and Weinberg, 1981.)
Focal accumulation of AChE at ectopic endplates was further examined using an antiserum to bovine brain AChE which stains endplates in adult rat muscles (Sanes and Hall, 1979) and which binds AChE solubilized from rat muscles (data not shown). The results agreed with those obtained by the histochemical method. No staining was detected at ectopic endplates up to 6 days after cutting the original nerve (Figs. 6A, B). At 8 days, fewer than half of the endplates stained faintly with anti-AChE (Figs. 6C, D). By 16 days most endplates were clearly stained and showed the broad thick staining that is characteristic of normal adult endplates (Figs. 6E, F). We then attempted to correlate the appearance of focal accumulations of AChE with the molecular forms of the enzyme present in the ectopically innervated region of the muscle. Bon et al., (1979) described six forms of mammalian AChE by their sedimentation coefficients. We have adopted their nomenclature for the peaks of activity on sedimentation gradients since we find the same peaks. There are three globular (G) forms, G1 (4 S), Gz (6 S), and Gq (10 S) which, by analogy with forms found earlier in electric organs of Electrophorus (Bon et al., 1976), are thought to consist of oligomers of a single catalytic subunit (the subscript is the number of catalytic subunits); and three asymmetric (A) forms: A4 (9 S), A8 (13 S), and AI2 (16 S) that are thought to contain a long, collagen-like tail with one, two or three of the globular tetramers attached to form the head. Measurements of the areas of peaks corresponding to G1 (the catalytic monomer) and Arz (the endplatespecific form) in 10 experiments are combined in Fig. 7 and velocity sedimentation gradients from one experiment are shown in Fig. 8. No Al2 was detected at 3 days at which time the AChE sedimentation profiles of ectopically innervated regions were indistinguishable from those of endplate-free regions of denervated muscles lacking a foreign nerve. Between Days 3 and 5,
WEINBERG
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Rat Neuromuscular
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FIG. 5. Morphology of ectopic endplates. Longitudinal sections of ectopically innervated soleus muscles were stained for AChE and impregnated with silver to reveal nerve fibers. Ectopic endplates are shown 3 days (A), 5 days (B), 1 week (C), 2 weeks (D), and 1 month (E) after cutting the original nerve. A normal endplate is shown in (F). Bar = 25 /.rrn.
there was a large increase in G1, and a small amount of Alz appeared. There was a three- to four-fold increase in Alz between Days 5 and 7. Also, the amount of As doubled and the amount of G1 continued to increase. Over the next few weeks the amount of AI2 more than doubled again. G1 and A8 also increased although to a lesser extent, while the amount of G4 remained nearly constant throughout the period of new endplate formation. Thus, there is good agreement among the various methods we used to detect AChE. Between 3 and 5 days after cutting the original nerve, histochemically detectable AChE appears at sites that may be intracellular, and the level of G1, which is thought to be a pre-
cursor of the larger forms (Rieger et al., 1976; Vigny et al., 1976), rises. Focal accumulations of AChE are first detected at ectopic endplates 2-3 days later, by both histochemical and immunochemical methods, coincident with a large increase in the amount of Au+ Histochemical, immunochemical, and biochemical analysis all show further, gradual increases in AChE as the ectopic endplates mature. Synaptic
Basal Lam&a
A layer of basal lamina completely ensheaths each adult muscle fiber, extending through the synaptic cleft and into the postjunctional folds. Recent experiments
262
FIG. 6. Accumulation stained with anti-AChE and rhodamine (right) Bar = 20 Km.
DEVELOPMENTAL
BIOLOGY
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of AChE at ectopic endplates shown by immunofluorescence. Cross-sections of ectopieally innervated muscles were followed by fluorescein-second antibody and rhodamine-bungarotoxin. Each field is shown under fluorescein (left) optics. Ectopic endplates are shown 6 days (A, B), 8 days (C, D), and 16 days (E, F) after cutting the original nerve.
have shown that the synaptic portion of the basal lamina is specialized at adult NMJs (Sanes and Hall, 1979) and that during reinnervation of muscles (Sanes et CL, 1978) or regeneration of damaged muscles (Burden et al., 1979), the synaptic basal lamina plays a major role in guiding the differentiation of the pre- and postsynaptic membranes. To see if, and when, the basal lamina at ectopic synapses becomes specialized, we examined developing ectopic endplates with two antisera that recognize components of synaptic basal lamina (Sanes and Hall, 1979). One was made against a muscle basement membrane collagen fraction and then adsorbed with endplate-free regions of muscle; the other was made against bovine anterior lens capsule. Both sera contain antibodies to synaptic portions of adult muscle fibers’
basal lamina sheath; antilens capsule also stains blood vessels and the perineurial sheath surrounding nerves. These two sera recognize determinants in synaptic basal lamina that are distinct from each other and from AChE (Sanes and Hall, 1979). When newly formed ectopic endplates were examined 31 a.+Not
4
denewated
1 b.+Five
FIG. 7. Summary of changes in activity of G,, the catalytic subunit monomer of AChE, and Am the endplate form of AChE. Activities were determined by measuring the areas under peaks on velocity sedimentation gradients (see Fig. 8) of ectopic endplate-containing regions of soleus muscles. Activities are shown as a function of time after denervation; each point represents a single experiment.
~
t c. One week
Fraction
DAYS AFTER 0ENER”AwJN
days
number
FIG. 8. Molecular forms of AChE at ectopic endplates. The region of a soleus muscle containing newly formed ectopic endplates was homogenized, run on a sucrose density gradient, and the fractions were assayed for AChE activity. Sedimentation coefficients for peaks were as follows: Gr, 3.8-4.1 S; G,, 10.0-10.3 S; As, 12.6-13.1 S; A12 15.816.6 S. Peaks corresponding to G2 and Ad are small and poorly resolved under these conditions. Gradients are shown of ectopically innervated regions 0 days (A), 5 days (B), 1 week (C), and 3 weeks (D) after cutting the original nerve. Sedimentation runs from left to right.
263
FIG. 9. Accumulation of muscles were stained with Each field is shown under (E, F), and 30 days (G, H)
synaptic basal lamina at ectopic endplates shown by immunofluorescence. Cross-sections of ectopically innervated adsorbed antibasement membrane collagen followed by fluorescein-second antibody and rhodamine-bungarotoxin. fluorescein (left) and rhodamine (right) optics. Ectopic endplates are shown 3 days (A, B), 6 days (C, D), 8 days after cutting the original nerve. Bar = 20 pm.
with the adsorbed antibasement membrane collagen serum no binding could be detected for the first 4 days after denervation (Figs. 9A, B). At 6 days faint, thin lines of staining were found at some of the receptor clusters (Figs. 9C, D). Most of these endplates were still visible even when the fluorescein-second antibody had been diluted 1:8 with unconjugated-second antibody (see Methods); therefore, this staining demonstrates a substantial accumulation of the synapse-specific antigens between Days 4 and 6. About half of the clusters stained with this antiserum at 8 days, while other clusters in the same section were not detectably stained (Figs. 9E, F). By 2 weeks after denervation almost all of the ectopic endplates were stained by this antiserum, and at 1 month after denervation (Figs. 9G, H) virtually all the endplates appeared as thick bands similar to normal endplates. At some older ectopic synapses (Figs. 9G, H), as at normal adult endplates (Sanes and Hall, 1979), the staining with adsorbed antibasement membrane collagen serum extended slightly beyond the borders of the rhodamine-BuTx-stained patch of AChRs. The results of staining with the antilens capsule serum were generally similar to those with the anti-
basement membrane serum: some endplates were stained on Day 6, about half were stained on Day 8, and nearly all were stained by 2 weeks. The initially thin line of staining became thicker by 1 month; however, unlike the adsorbed antibasement membrane collagen serum, the staining with antilens capsule was always coextensive with the AChR clusters. There may have been some staining 4 days after denervation since endplates were always among the brightest regions of the fiber surface; however, since the entire surface of soleus muscle fibers, unlike diaphragm muscle fibers (Sanes and Hall, 1979), was lightly stained by antilens capsule, it was difficult to determine if endplates were specifically stained at 4 days. In summary, the basal lamina of adult muscle fibers becomes specialized during the formation of new synaptic sites, Synapse-specific antigens are first detectable at some ectopic synapses by 6 days after denervation, and they accumulate gradually over the next few weeks until all the endplates appear mature. We have also found that cutting the foreign nerve on Day 4 prevents the accumulation of the synaptic basal lamina antigens at ectopic endplates (data not shown).
DEVELOPMENTALBIOLOGY
264 DISCUSSION
We have studied the formation and maturation of new, ectopic, neuromuscular synapses between an implanted foreign nerve and soleus muscle fibers in adult rats. We focused on the differentiation of four molecular components of the synapse: AChRs which are integral membrane proteins concentrated at the crests of the subsynaptic folds; AChE which is concentrated at the synapse, and at least some of which is associated with synaptic basal lamina; and two immunologically defined components of the synaptic basal lamina. We find that all of these components accumulate at ectopic synapses in less than a month after the original nerve is cut. AChRs cluster before specialized basal lamina components including AChE accumulate in detectable amounts. Clustered AChRs become densely packed and metabolically stable before AChE accumulates. Synapse-specific components of the basal lamina accumulate concurrently with or just before accumulation of AChE. The first detectable event occurs 2 days after the original nerve is cut, when clusters of AChRs appear underneath the ectopic nerve. In electrophysiological studies of ectopic synapse formation on rat soleus muscles, Ldmo and Slater (1976, 1978) found that the earliest signs of transmission occurred at 2 l/2-3 days. Receptors in the first clusters to be detected have a moderately high density, about half of their final density. The final density is achieved within a few days after the clusters form. This change may correspond to the transition from “aggregates” to “plaques” of AChRs recently described by Steinbach (1981) and the development of resistance to dispersal by low Ca2+ recently described by Bloch and Steinbach (1981) during synapse formation in developing muscle. The rate of degradation of clustered AChRs in rat muscle decreases 5- to lo-fold within the first few days after clustering at both ectopic and embryonic NMJs (Reiness and Weinberg, 1981). The accumulation of AChE was studied immunocytochemically, histochemically, and by sedimentation analysis. All three methods first detected a high concentration of synaptic AChE at the ectopic endplates around 1 week after cutting the original nerve, and the amount continued to increase over the next 2 weeks. Two components of the NMJ, that in adult muscle are associated with the basal lamina (Sanes and Hall, 1979), also occur at mature ectopic NMJs. Synapse-specific antigens were detected at some endplates as early as Day 6; the fraction of endplates containing these antigens increased during the next week until by 2 weeks they were present at virtually all endplates. Because
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ectopic NMJs form around a preexisting basal lamina that is initially extrasynaptic, a process of remodeling must occur in the extracellular matrix (as well as in the muscle’s plasma membrane) as the synaptic matures. At synapses formed during normal development (Kelly and Zacks, 1969; Jacob and Lentz, 1979) and in cell culture (Frank and Fischbach, 1979), the basal lamina appears between the nerve terminal and the muscle fiber at a very early stage. Whether this structure contains the synaptic antigens from the beginning of whether some form of remodeling of the basal lamina takes place during development is not yet known. The earliest times at which AChR clusters (Bevan and Steinbach; 1977; Braithwaite and Harris, 1979), accumulation of AChE at synaptic sites (Bennett and Pettigrew, 1974), A1z, the endplate form of AChE (Vigny et al., 1976), and basal lamina in the synaptic cleft (Kelly and Zacks, 1969) have been reported in rat embryos are all around the 15th or 16th days of gestation. Thus, the sequence of steps in synapse formation is difficult to resolve in the embryo, although it seems likely that AChE accumulates after receptors cluster (Rubin et al., 1979; Kullberg et al., 1980). Our observations at newly formed ectopic NMJs clearly separate the clustering of AChRs from the focal accumulation of AChE at synapses. Furthermore, in contrast to the results in rat embryos Reiness and Weinberg, (see 1981), AChR density and metabolic turnover time reached nearly mature values at ectopic synapses before detectable amounts of AChE had accumulated. Our results also suggest that the remodeling of the basal lamina when new synapses are formed on adult muscle fibers is concurrent with or slightly precedes the accumulation of AChE, which is associated with the basal lamina at adult NMJs (Hall and Kelly, 1971; Betz and Sakmann, 1974; McMahan et al., 1978). The mechanisms by which the postsynaptic membrane and the synaptic basal lamina are assembled are unknown; however, our results show that they are not formed as a unit. Recent experiments have shown that components of the synaptic basal lamina play important roles in the differentiation of regenerating axons into nerve terminals when the original synaptic sites are reinnervated (Sanes et al., 1979) and in the clustering of AChRs when damaged muscle regenerates (Burden et aZ., 1979). It is an attractive possibility that during the formation of new NMJs, components of the basal lamina may play a role in directing the specialization of other components of the synapse. Our experiments clearly show that complete differentiation of the basal lamina at newly forming ectopic synapses takes place well after functional transmission is established and the initial clustering of receptors has occurred. However, our exper-
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iments provide no evidence on whether unknown antigens or low levels of known antigens play a role in the formation of the NMJ, or whether components of the synaptic basal lamina regulate later steps in the maturation of pre- and postsynaptic membranes. We thank Miu Lam for expert technical assistance, and Eric Frank, who first demonstrated the procedure for forming ectopic endplates to us. This work was supported by grants from the Muscular Dystrophy Association (MDA), the National Institutes of Health (NIH), and the National Science Foundation to Z.W.H. C.B.W. was supported by an NIH training grant and J.R.S. by a postdoctoral fellowship from MDA REFERENCES AITKEN, J. T. (1950). Growth of nerve implants in voluntary muscle. J. Anat. 34, 38-49. BEERMAN, D. H., and CASSENS, R. G. (1976). A combined silver and acetylcholinesterase method for staining intramuscular innervation. Stain Technol. 51.173-177. BENNETT, M. R., and PETTIGREW, A. G. (1974). The formation of synapses in striated muscle during development. J. Physiol. 241, 515545. BENNETT, M. R., and PETTIGREW, A. G. (1975). The formation of neuromuscular synapses. Cold Spring Harbor Symp. Quant. BioL 49, 409-424. BET& W., and SAKMANN, B. (1973). Effects of proteolytic enzymes on function and structure of frog neuromuscular junctions. J. Physiol. 230,673-688. BEVAN, S., and STEINBACH, J. H. (1977). The distribution of a-bungarotoxin binding sites on mammalian skeletal muscle developing in vivo. J. Physiol. 267, 195-213. BLOCH, R. J., and STEINBACH, J. H. (1981). Reversible disruption of acetylcholine receptor clusters at the developing rat neuromuscular junction. Develop. BioL 81, 386. BON, S., HUET, M., LEMONNIER, M., RIEGER, F., and MASSOULIE, J. (1976). Molecular forms of Electrophorus acetylcholinesterase. Eur. J. Biochem. 68,523-530. BON, S., VIGNY, M., and MASSOULIE, J. (1979). Asymmetric and globular forms of acetylcholinesterase in mammals and birds. Proc. Nat. Acad Sci. USA 76,2546-2550. BRAITHWAITE, A. W., and HARRIS, A. J. (1979). Neural influence on acetylcholine receptor during embryonic development of skeletal muscles. Nature (London) 279, 549-551. BURDEN, S. (1977). Development of the neuromuscular junction in the chick embryo: the number, distribution, and stability of acetylcholine receptors. Develop. Biol. 57,317-329. BURDEN, S. J., SARGENT, P. B., and MCMAHAN, U. J. (1979). Acetylcholine receptors in regenerating muscle accumulate at original synaptic sites in the absence of the nerve. J. Cell BioL 82,412-425. EDWARDS, C. (1979). The effects of innervation on the properties of acetylcholine receptors. Neuroscience 4, 565-584. ELMAN, G. L., COURTNEY, K. D., ANDRES, V., and FEATHERSTONE, R. M. (1961). A new and rapid calorimetric determination of acetylcholinesterase activity. Biochem. Pharmacology 7, 88-95. ELSBERG, C. A. (1917). Experiments on motor nerve regeneration and the direct neurotization of paralyzed muscles by their own and foreign nerves. Science 45, 318-320. FEX, S., SONNESSON,B. THESLEFF, S., and ZELENA, J. (1966). Nerve implants in botulinum poisoned mammalian muscle. J. Phusiol. 184, 872-882.
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FEX, S., and THESLEFF, S. (1967). The time required for innervation of denervated muscles by nerve implants. Life Sci. 6,635-639. FRANK, E., and FISCHBACH, G. D. (1979). Early events in neuromuscular junction formation in vitro. J. Cell Biol. 83, 143-158. FRANK, E., GAUTVIK, K., and SOMMERCHILD, H. (1975). Cholinergic receptors at denervated mammalian motor end-plates. Acta. PhpsioL Stand. 95, 66-76. GREENBERG, A. J., PARKER, K. K., and TREVOR, A. J. (1977). Immunochemical studies of mammalian brain acetylcholinesterase. J. Neurochem. 29,911-917. GUTH, L., and ZALEWSKI, A. A. (1963). Disposition of cholinesterase following implantation of nerve into innervated and denervated muscle. Exp. Neurol. 7.316-326. GUTMANN, E., and HANZLIKOVA, V. (1967). Effects of accessory nerve supply to muscle achieved by implantation into muscle during regeneration of its nerve. Physiologia Bohemoslovaca 16,244-250. GIJTMANN, E., and YOUNG, J. Z. (1944). The re-innervation of muscle after various periods of atrophy. J. Anat. 78.15-43. GWYN, D. G., and AITKEN, J. T. (1964). New motor end-plates and their relationship to muscle fibre injury. Nature (London) 203,651652. GWYN, D. G., and AITKEN, J. T. (1966). The formation of new motor endplates in mammalian skeletal muscle. J Anat. 100, 111-126. HALL, Z. W. (1973). Multiple forms of acetylcholinesterase and their distribution in endplate and non-endplate regions of rat diaphragm muscle. J. Neurobiol. 4, 343-361. HALL, Z. W., and KELLY, R. B. (1971). Enzymatic detachment of endplate acetylcholinesterase from muscle. Nature New BioL 232,6263. JACOB, M., and LENTZ, T. L. (1979). Localization of acetylcholine receptors by means of horseradish peroxidase-a-bungarotoxin during formation and development of the neuromuscular junction in the chick embryo. J. Cell Biol. 82, 195-211. JANSEN, J. K. S., LIMO, T., NICOLAYSEN, K., and WESTGAARD, R. H. (1973). Hyperinnervation of skeletal muscle fibers: Dependence on muscle activity. Science 181, 559-561. KELLY, A. M., and ZACKS, S. I. (1969). The fine structure of motor endplate morphogenesis. J. Cell BioL 42.154-169. Ko, P. K., ANDERSON, M. J., and COHEN, M. W. (1977). Denervated skeletal muscle fibers develop discrete patches of high acetylcholine receptor density. Science 196.540-542. KOENIG, J. (1971). Contribution a l’etude de la neoformation experimentale des plaques matrices de rat. Arch. Anat. Microscop. MOTphol. Exp. 60, l-26. KORNELIUSSEN, H., and SOMMERSCHILD, H. (1976). Ultrastructure of the new neuromuscular junctions formed during reinnervation of rat soleus muscle by a “foreign” nerve. Cell Tiss. Res. 167.439-452. KUFFLER, D., THOMPSON, W., and JANSEN, J. K. S. (1977). The elimination of synapses in multiply innervated skeletal muscle fibres of the rat: Dependence on distance between endplates. Brain Res. 138.353-358. KULLBERG, R. W., MIKELBERG, F. S., and COHEN, M. W. (1980). Contribution of cholinesterase to developmental decreases in the time course of synaptic potentials at amphibian neuromuscular junctions. Develop. Biol. 75, 255-267. LENTZ, T. L. (1969). Development of neuromuscular junctions in regenerating limbs of the newt Triturus. J. Cell Biol. 42.431-443. LIMO, T., and SLATER, C. R. (1976). Control of neuromuscular synapse formation. In “Synaptogenesis” (L. Taut, ed.), pp. 9-30. Naturalia & Biologia, Jouy en Josas. LIMO, T., and SLATER, C. R. (1978). Control of acetylcholine sensitivity and synapse formation by muscle activity. J. PhysioL 275,391-402. MCMAHAN, U. J., SANES, J. R., and MARSHALL, L. M. (1978). Cholin-
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esterase is associated with the basal lamina at the neuromuscular junction. Nature (London) 271, 172-174. MILEDI, R. (1963). Formation of extra nerve-muscle junctions in innervated muscle. Nature (London) 199.1191-1192. REINESS, C. G. and WEINBERG, C. B. (1981). Metabolic stabilization of aeetylcholine receptors at newly formed neuromuscular junctions in rat. Develop. Biol. 83, 247-254. RIEGER, F., FAIVRE-BAUMAN, A., BENDA, P., and VIGNY, M. (1976). Molecular forms of acetylcholinesterase: Their de nowo synthesis in mouse neuroblastoma cells. J. Neurochem. 27,1059-1063. RUBIN, L. L., SCHUETZE, S. M., and FISCHBACH, G. D. (19’79). Aeeumulation of acetylcholinesterase at newly formed nerve-muscle synapses. Develop. Biol. 69, 46-58. SANE&J. R., and HALL, 2. W. (1979). Antibodies that bind specifically to synaptic sites on muscle fiber basal lamina. J. Cell Biol. 83, 357370. SANES, J. R., MARSHALL, L. M., and MCMAHAN, U. J. (1978). Reinnervation of muscle fiber basal lamina after removal of myofibers. Differentiation of regenerating axons at original synaptic sites. J. Cell Biol. 78, 176-198.
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STEINBACH, J. H. (1981). Developmental changes in acetylcholine receptor aggregates at rat skeletal muscle neuromuscular junctions. Develop. Biol 84, 267-276. VIGNY, M., KOENIG, J., and RIEGER, F. (1976). The motor end-plate specific form of acetylcholinesterase: Appearance during embryogenesis and re-innervation of rat muscle. J. Neurochem. 27,13471353. WAERHAUG, O., KORNELIUSSEN, H., and SOMMERSCHILD, H. (1977). Morphology of motor nerve terminals on rat soleus muscle fibers reinnervated by the original and by a “foreign” nerve. Anat. Embrgol. 151, 1-15. WAKE, K. (1976). Formation of myoneural and myotendinous junctions in the chick embryo. Cell Tiss. Res. 173,383-400. WEINBERG, C. B., and HALL, Z. W. (1979a). Junctional form of acetylcholinesterase restored at nerve-free endplates. Develop. Biol. 68, 631-635. WEINBERG, C. B., and HALL, Z. W. (197913). Antibodies from patients with myasthenia gravis recognize determinants unique to extrajunctional acetylcholine receptors. Proc. Nat. Acad. Sci. USA 76, 504-508.
BIOLOGY
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(1981)
Formation of Neuromuscular Junctions in Adult Rats: Accumulation of Acetylcholine Receptors, Acetylcholinesterase, and Components of Synaptic Basal Lamina CRISPIN B. WEINBERG, Neurobiology
JOSHUA R. SANES,’ AND ZACH W. HALL
Division, Department of Physiology, University of Califomzia School of Medicine, San Francisco, California Department of Neurobiology, Harvard Medical School, 25 Shattuck St., Boston, Massachusetts 02115
9/l&?,
and
Received August 21, 1980; accepted in revised form November 17, 1980 We describe the appearance and accumulation of four specialized molecular components of the adult neuromuscular junction during ectopic endplate formation in adult rat soleus muscles. One component, the acetylcholine receptor (AChR), is a major constituent of the postsynaptic muscle membrane. The other three, acetylcholinesterase (AChE) and two extracellular synapse-specific antigens, are associated, at least in part, with the basal lamina in the synaptic cleft. The accumulation of each component was studied by immunocytochemistry. In addition, the accumulation of AChRs was measured by autoradiography after reaction with ‘%I-a-bungarotoxin, and AChE was examined by histoehemistry and by sedimentation analysis. Endplate formation was initiated by cutting the original nerve to a muscle in which a foreign nerve had been previously implanted. Within 2 days, clusters of AChRs appeared in the new endplate zone. The density of AChRs in these clusters increased from 1 X lo4 sites/pm’ to nearly the final value of 2 X 10’ sites/ pm2 by 4 days. The cluster continued to grow in size and receptor number over the next month. AChE was not detected on the surface of the muscle fiber until after 1 week, when it was present at some, but not all ectopic endplates. Its appearance coincided with a rapid accumulation of the endplate-specific, 16 S form of AChE (A& in the portion of the muscle containing new endplates. By 2 weeks virtually all endplates stained for AChE and by 1 month both immunochemical and histochemical staining resembled that of normal adult endplates. The synapse-specific basal lamina antigens that we studied were detected at some endplates by Day 6, and their further appearance followed a time course similar to, or slightly ahead of, that of AChE. Thus maturation of the synaptic basal lamina occurs after the AChRs have formed clusters and achieved nearly their final density.
INTRODUCTION
are assembled and because the synaptic basal lamina has been shown to influence the differentiation of preand postsynaptic membranes during regeneration of damaged nerve and muscle fibers (Sanes et al., 1978; Burden et al., 1979). We wished to examine the temporal relation between the assembly and macromolecular differentiation of the synaptic basal lamina and of the postsynaptic muscle membrane during the formation of the NMJ. We chose to study the ectopic synapses that are formed between transplanted “foreign” motor axons and denervated soleus muscle fibers in the adult rat. During spontaneous reinnervation, motor axons preferentially reinnervate muscle fibers at original synaptic sites (Gutmann and Young, 1944; for review see Bennett and Pettigrew, 1975). In contrast, when a nerve is implanted far from the original endplates, and the original innervation is interrupted, new endplates are formed at ectopic sites on previously nonsynaptic portions of the muscle fiber surface (Elsberg, 1917; Aitken, 1950; Guth and Zalewski, 1963; Miledi, 1963; Fex et al., 1966; Jansen et al., 1973). In this case, specializations of the postsynaptic muscle membrane and of the synaptic cleft are formed de novo. The physiological (Jan-
At the adult neuromuscular junction (NMJ), both the postsynaptic muscle membrane and the extracellular basal lamina in the synaptic cleft are biochemically specialized. Acetylcholine receptors (AChRs) are clustered in the postsynaptic membrane, and acetylcholinesterase (AChE) is highly concentrated in the synaptic cleft where at least some of it is associated with the basal lamina (Hall and Kelly, 1971; Betz and Sakmann, 1973; McMahan et al., 1978). In rat muscle, a particular form of AChE (16 S) appears to be specifically associated with endplates (Hall, 1973; Vigny et al., 1976). Several other components of the basal lamina have been shown by immunological methods also to occur at high density in the synaptic cleft, but not elsewhere on the muscle fiber’s surface (Sanes and Hall, 1979). The relation between the differentiation of the basal lamina and of the postsynaptic membrane during synapse formation is of interest both in terms of understanding how specialized components of the cell surface ’ Present address: Department of Physiology and Biophysics, ington University School of Medicine, St. Louis, MO. 63110
Wash-
255 0012-1606/81/080255-12$02.00/O Copyright All rights
0 1981 by Academic Press, Inc. of reproduction in any form reserved.
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sen et al., 1973; Frank et al., 1975; LQmo and Slater, 1976,19’78) and morphological (Korneliussen and Sommerschild, 1976; Waerhaug et al., 1977) events that occur during the formation of ectopic NMJs in the rat soleus muscle have been well described and form a useful framework in which to investigate molecular aspects of synaptogenesis. We describe here the accumulation and differentiation of AChR, AChE, and two synaptic basal lamina antigens during the formation of ectopic NMJs in adult rats. Our principal finding is that the differentiation of the postsynaptic muscle membrane and of the synaptic basal lamina do not occur together but follow different time courses. METHODS
Ectopic Endplates. Ectopic innervation of rat soleus muscles was produced as described by Frank et al. (1975). Sprague-Dawley rats (100-200 g) were anesthetized with sodium pentobarbital and the superficial fibular nerve was dissected free in the lower leg and cut at the ankle. The cut end of the nerve was placed on the proximal portion of the soleus muscle, at least 5 mm away from the original endplates, which occupy a band in the middle of the muscle. The overlying muscle was then sutured and the incision was closed with wound clips. To initiate synaptogenesis, the soleus muscle was denervated by removing a 5- to lo-mm segment of the tibia1 nerve 2 to 4 weeks later (= Day 0). At appropriate intervals thereafter, animals were killed and their soleus muscles removed for histochemical or biochemical analysis. In some cases, to monitor the progress of ectopic innervation, the muscles were dissected in oxygenated Kreb’s solution, the foreign nerve was stimulated with a suction electrode, and tension was measured with a Grass force transducer. Ectopic innervation failed to develop in about 10% of the animals. Histology. Nerve fibers and sites of AChE activity were examined in lOO-pm longitudinal cryostat sections of the entire soleus muscle. The sections were stained by combined cholinesterase histochemistry and silver impregnation of nerve fibers (Beerman and Cassens, 1976). Immunocytochemistry. The accumulation of synapsespecific molecules was studied immunocytochemically, using antisera to rat skeletal muscle AChR (Weinberg and Hall, 1979b), bovine brain AChE (Greenberg et al., 1977; kindly provided by Drs. A. Greenberg and A. Trevor of UCSF), bovine lens capsule (Sanes and Hall, 1979), and a basement membrane collagen-rich fraction from rat muscle (Sanes and Hall, 1979). The antibasement membrane collagen was adsorbed before use with
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connective tissue from endplate-free regions of rat diaphragm, in order to reveal synapse-specific antibodies (Sanes and Hall, 1979); the other sera was not adsorbed before use. Anti-AChR was used at a dilution of 1:500 in phosphate-buffered saline (PBS) containing 10 mg/ ml BSA, while other sera were diluted 1:200. Immunocytochemical methods have been detailed by Sanes and Hall (1979). Briefly, cross-sections of frozen, unfixed muscle were cut in a cryostat, incubated first with antiserum and then with a mixture of fluorescein-conjugated goat anti-rabbit IgG (GAR, Cappel) and rhodamine-a-bungarotoxin (rhodamine-BuTx, a gift from Drs. Peter Ravdin and Darwin Berg of University of California, San Diego), and finally viewed through selective filters that allowed us to see fluorescein and rhodamine separately. The rhodamine-BuTx permitted us to verify the identity of antibody-stained endplates, and also provided an additional way of assessing the distribution of AChRs at ectopic synapses. Although immunocytochemical studies of developing synapses do not provide a quantitative measure of antigen concentrations, we desired to know if the time at which we first detected synapse-specific antigens was determined entirely by the sensitivity of our method (as would occur if the antigen concentration rose gradually until it finally reached the threshold for detection) or whether the antigen was present at a concentration well above the sensitivity of our method on the first day we could detect it (in which case the “appearance” of an antigen would represent a more or less abrupt rise in its concentration). To distinguish between these two possibilities, we performed an experiment using, as second antibody, mixtures of fluorescein-conjugated and unconjugated GAR in varying proportions with the total titer of antibody kept constant. The nonspecific background staining is reduced along with the specific staining, as the concentration of fluorescein is decreased. Thus the precise value of the dilution of fluorescein-GAR that gives just-visible staining is not significant; however, this test can show whether antigens are present in concentrations that are well above the limit of detection. At adult endplates, binding of antibodies to antigens in the synaptic basal lamina was easily detected when a 1:8 mixture of fluorescein-conjugated GAR and unconjugated GAR was substituted for the usual amount of fluorescein GAR. Autoradiography. AChRs were quantitated by autoradiography of single muscle fibers labeled with ‘%ICX-BuTx as described previously (Reiness and Weinberg, 1981). A nearly saturating dose of 1251-~-B~T~ (lo-12 cLg/lOOg body wt) was injected into the femoral artery of an anesthetized rat. Three hours after labeling, the soleus muscle was removed, fixed, and reduced to single fibers in a VirTis homogenizer. The fibers were dried
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onto gelatin-coated slides which were then dipped in Kodak NTB-2 emulsion, exposed at 4”C, developed, and examined under dark-field illumination. Silver grains in each cluster were counted and the area of the cluster was measured. The number of toxin binding sites was calculated assuming that 20% of the ‘=I emissions produced a grain (Burden, 1977). For normal soleus endplates we obtained values of 1.4 +- 0.2 X 10’ receptors per endplate and a density of 2.7 ? 0.2 X lo* receptors/ pm2 (mean f SEM, n = 6) which are in good agreement with values obtained in other rat skeletal muscles (reviewed in Edwards, 1979). In one control experiment we visualized the same endplates by both autoradiography and immunocytochemistry. Muscles that had been labeled with ‘25I-aBuTx in vivo were dissected, fixed lightly (4% formaldehyde for 10 min at room temperature), reduced to single fibers, and stained with anti-AChR. After endplates were located and photographed, the muscle fibers were subjected to autoradiography. Five endplates were later relocated and photographed under dark-field illumination. The images obtained by the two techniques were superimposable and the regions of most intense fluorescence generally corresponded to regions of highest grain density (Fig. 1). Sedimentation analysis of acetylcholinesterase. The forms of AChE were separated by sedimentation on sucrose gradients as described previously (Hall, 1973; Weinberg and Hall, 1979a). The ectopically innervated region of the soleus was separated from the region containing the original endplates, homogenized in ice-cold buffer (1 M NaCl, 1% Triton-X-100, 50 mM Tris-HCl, and 0.2 mM EDTA, pH 7.4), and centrifuged at 15,000g for 15 min at 4°C. Aliquots of the supernatant were run on linear sucrose gradients (5-20s). AChE activity was assayed by the method of Ellman et al. (1961) and under
FIG. 1. Correspondence of immunocytochemistry raphy. The same endplate on a single muscle immunocytochemical staining with anti-AChR (B). Bar = 20 Wm. radiography of ‘251-~-B~T~
and autoradiogfiber is shown after (A) and after auto-
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the conditions used was directly proportional to the enzyme concentration. Control experiments with inhibitors of specific and nonspecific cholinesterases, (BW234c51) and tetramonoisopropyl pyrophosphortetramide (iso-OMPA), respectively, showed that nonspecific cholinesterases accounted for less than 15% of the activity in each peak on gradients of these extracts. RESULTS
Development
of Ectopic
Endplates
Ectopic NMJs were produced on denervated soleus muscle fibers by axons of a foreign nerve, the fibular nerve. In a first operation, the fibular nerve was implanted on an endplate-free zone of the soleus. Two to four weeks later, ectopic synapse formation was initiated by cutting the original nerve (= Day 0). Ectopic synapses are more numerous and develop more quickly when this schedule is used than when a foreign nerve is implanted on a muscle at the same time that the original nerve is cut (Fex and Thesleff, 1967). Neither structural nor functional signs of ectopic synapse formation were seen in muscles before the original nerve was cut (see also Elsberg, 1917; Aitken, 1950; Guth and Zalewski, 1963). In silver-stained sections of normally innervated muscles with an implanted foreign nerve, foreign nerve fibers were seen growing along muscle fibers, but these axons rarely branced and had few growth cones (see also Aitken, 1950). No contractions were recorded from such muscles when the foreign nerve was stimulated, and accumulations of AChRs or AChE were generally not seen on soleus muscle fibers in the region of the fibular nerve implant. One control muscle did contain a few endplates on fibers that lay directly under the foreign nerve; these synapses were presumably formed on muscle fibers that had been injured during the implantation operation and thus rendered susceptible to hyperinnervation (Miledi, 1963, Gwyn and Aitken, 1964). Ectopic synapses formed quickly once the original nerve was cut. Three days after denervation, implanted muscles had larger numbers of highly branched foreign nerve fibers with many growth cones (see, e.g., Fig. 5A). Korneliussen and Sommerschild (1976) have described vesicle-laden terminal boutons closely apposed to muscle fibers at 3-5 days. We first saw contraction of a few muscle fibers in response to stimulation of the foreign nerve on Day 3. This is consistent with the observation that spontaneous and evoked endplate potentials are first seen 2 l/2 to 3 days after denervation (Lomo and Slater, 1976,1978; see also Fex and Thesleff, 1967). Thus morphological and physiological evidence of ectopic synapse formation is present within 3 days of denervation.
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The average strength of contractions evoked by stimulating the foreign nerve increased gradually over the next 2-3 months (see also Fex and Thesleff, 1967; Frank et al., 1975). Foreign nerve terminals visualized by silver impregnation arborized and developed more extensive contacts with muscle fibers during this period. Subsynaptic folds have been reported at 10 days and become more extensive over the next 2 months (Korneliussen and Sommerschild, 1976). Ectopic endplates resemble normal adult endplates both morphologically and physiologically by 2-3 months. Acetylcholine
Receptors
When cross-sections of normal soleus muscle were stained with anti-AChR and fluorescein-GAR or with rhodamine-BuTx, the endplates appeared as bright broad bands or groups of spots (Sanes and Hall, 1979). The patterns of staining observed with rhodamineBuTx and with antiserum to AChR were identical, while muscles treated with preimmune sera and fluoresceinGAR showed little fluorescence above background. Only AChRs clustered at high density were revealed by these methods; extrajunctional receptors in embryonic or denervated adult muscles were not seen. In ectopically innervated soleus muscles, AChR clusters were first seen at 2 days. They appeared as faint, poorly defined patches of fluorescence on the muscle fiber surface that were coincident when seen under fluorescein and rhodamine optics (Figs. 2A, B), and were found only in the region of the soleus underlying the
FIG. 2. Accumulation of AChRs at ectopic endplates shown were stained with anti-AChR followed by fluorescein-second fluorescein optics. (B) is the same field as (A) photographed 6 days (D), and 16 days (E) after cutting the original nerve.
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foreign nerve. These presumably correspond to the patches of high ACh sensitivity found under the foreign nerve at 2 l/2 to 3 days (LQmo and Slater, 1976,1978). Because the staining of newly formed clusters was faint, the combined antibody and toxin labeling gave much greater sensitivity than either method alone; spots stained lightly, and presumably nonspecifically, by only one reagent or the other were found throughout the muscle. The AChR clusters seen with fluorescent antibodies and rhodamine-BuTx were clearly different from the “hot spots” that appear in extrajunctional regions of muscles several days after denervation (Ko et al., 1977). In rat soleus muscles the hot spots are typically 1 Mm2 and are almost never larger than 20 lrn2 (Ko et al., 1977), while by 2-3 days, the clusters that we identify as ectopic endplates have areas of 160-210 pm2. Hot spots were seen in autoradiographs of ectopically innervated muscles but were much rarer than in control (nonimplanted) denervated muscles. In ectopically innervated muscles, they were almost never seen more than 10 days after cutting the original nerve; this suggests that they were suppressed, as are unclustered extrajunctional AChRs (L6mo and Slater, 1976, 1978), by innervation. Three to four days after denervation patches of AChRs were more frequently seen and appeared in cross-section as short thin lines (Fig. 2C). By 1 week the lines had become longer but remained thin (Fig. 2D). During the second week the ectopic clusters became brighter and thicker so that by 16 days they were sim-
by immunofluorescence. Cross-sections of ectopically innervated soleus muscles antibody and rhodamine-bungarotoxin. (A) and (c-F) were photographed with with rhodamine optics. Eetopie endplates are shown 2 days (A, B), 3 days (C), (Ff shows normal endplates. Bar = 20 pm.
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Adult Rat Neuromuscular
ilar in appearance to, but smaller than mature synapses. At 1 month (Fig. 2E) they were indistinguishable from normal endplates. The thickening of the lines of staining may correspond to the formation of subsynaptic folds which starts during the second and third weeks (Korneliussen and Sommerschild, 1976). Autoradiography of muscles labeled with lWI-cr-BuTx confirmed the results obtained with anti-AChR and rhodamine-BuTx, and enabled us to determine the number and density of AChRs at developing ectopic endplates. Autoradiographs of some ectopic endplates are shown in Fig. 3 and the data obtained from these experiments are summarized in Table 1. Small, low-density clusters were first seen as early as 2 days. The average number of AChRs in a cluster steadily increased over the next month to achieve a value close to that found in adult endplates. The AChR density nearly doubled between the second and fourth days, and thereafter increased only slightly (Table 1, Fig. 4A). Thus the total number of AChRs in each cluster increases largely through the expansion of the total area occupied by the cluster. In experiments reported in the accompanying paper, we have found that the rate at which clustered AChRs are degraded changes between 2 and 6 days after denervation from an initially rapid value to a slower one characteristic of AChRs at adult endplates (Reiness and Weinberg, 1981). The time course of this change is roughly similar to that of the AChR density and, for comparison, is shown in Fig. 4B. Thus two of the important characteristics of AChRs at adult endplates, their high density and their slow rate of metabolic turnover, are well established at ectopic endplates within a few days after clusters of AChRs first appear. Many of the muscle fiber segments (typically l-mm long) observed during the first week had more than one
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cluster of AChRs. The number of fibers with more than one cluster decreased over the next few weeks until, by 3 weeks, most fibers had only one receptor cluster. This decrease presumably corresponds to the decrease in multiple innervation at newly formed ectopic NMJs that has been observed using morphological and electrophysiological techniques (Aitken, 1950; Frank et al., 1975; Kuffler et al., 1977). In summary, AChRs accumulate at ectopic NMJs at the same time as, or slightly before, the onset of synaptic transmission. Within a few days the clustered receptors are as metabolically stable and as densely packed as AChRs at mature NMJs. The size of the clusters, however, continues to increase for several weeks.
Acetylcholinesterase Although AChE is present throughout muscle fibers, its concentration at the mature NMJ is at least lOOOfold greater than in extrasynaptic regions (Hall, 1973). In addition, in rat muscle, a particular form of the enzyme, distinguished by velocity sedimentation (the 16S form) is specificially associated with the endplate region (Hall, 1973; Vigny et al., 1976). Both histochemical staining for AChE (see also Guth and Zalewski, 1963; Gutman and Hanzlikova, 1967) and the endplate form of the enzyme (Vigny et al., 1976) have been described at mature ectopic synapses. We therefore examined the accumulation of AChE and its molecular forms during the development of ectopic synapses. No AChE reaction porduct could be seen associated with the foreign nerve in normally innervated soleus muscles (Guth and Zalewski, 1963) or during the first 4 days after cutting the original nerve (Fig. 5A). The first change in the staining pattern of ectopically in-
FIG. 3. Accumulation of AChRs at ectopic endplates shown by autoradiography. Muscles which had been labeled with ‘%I-a-BuTx reduced to single fibers and processed for autoradiography. Dark-field photomicrographs are shown of eetopic endplates 2 days (A), (B), and 40 days (C) after cutting the original nerve, and of a normal endplate (D). Bar = 20 pm.
were 8 days
260
DEVELOPMENTAL
TABLE ACETYLCHOLINE Days after denervation
Note.
Ectopic
endplates
2 3 4 6 12 17 40
Normal
endplates
0
Means
k SEM
(n = 3-8; usually
RECEPTORS
1.7 3.2 4.4 5.7 8.9 12.3 13.2
10
Days
20
* f f + f f k
34, 1981
1 AT ECTOPIC
0.1 0.4 0.2 0.2 0.4 1.0 1.5
x X x x x X X
ENDPLATES Density (AChRs/rm’)
Number of AChRs/cluster lo6 lo6 lo6 lo6 lo6 lo6 lo6
14.0 + 1.5 x lo6
1.1 1.5 1.7 1.5 1.9 2.2 2.0
k f + f + * f
0.1 0.2 0.3 0.2 0.1 0.2 0.2
Area (rm’) x x x x x x x
lo4 lo4 lo4 10’ lo4 10’ lo4
2.7 k 0.2 X lo4
160 210 260 380 470 560 660
+ + k f + k ct
15 20 30 60 30 40 50
520 ck 50
4-6).
nervated muscles was observed on Day 5 when the region of the muscle fiber underneath some nerve endings and growth cones was diffusely stained (Fig. 5B). This staining appeared to be cytoplasmic and may represent the accumulation of AChE in subsynaptic cisternae prior to its deposition on the muscle fiber surface (Lentz, 1969; Wake, 1976). The first focal staining for AChE under nerve endings was observed 1 week after denervation (Fig. 5C, Lbmo and Slater, 1976, 1978). Stained areas appeared as the single “ovoid cups” described by Koenig (1971). Two weeks after denervation the patches were darker and more sharply defined (Fig. 5D), and by l-2 months they formed groups of spots or ovoid cups (Fig. 5E) which resembled normal endplates (Fig. 5F). The increase in number of patches or ovoid cups at single ectopic endplate probably corresponds to the increase in the number of synaptic boutons described by Korneliussen and Sommerschild (1976) during the second and third weeks after cutting the original nerve.
0
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30
40
Normal
after denewation
FIG. 4. Density and turnover of AChRs at ectopic endplates. Ectopically innervated muscles were labeled with ‘Z51-a-BuTx in V&O and processed for autoradiography at various times after labeling. The density (A) and mean half-time for turnover (B) for clustered receptors are plotted as a function of the time after cutting the original nerve at which the muscle was labeled. (Turnover times from Reiness and Weinberg, 1981.)
Focal accumulation of AChE at ectopic endplates was further examined using an antiserum to bovine brain AChE which stains endplates in adult rat muscles (Sanes and Hall, 1979) and which binds AChE solubilized from rat muscles (data not shown). The results agreed with those obtained by the histochemical method. No staining was detected at ectopic endplates up to 6 days after cutting the original nerve (Figs. 6A, B). At 8 days, fewer than half of the endplates stained faintly with anti-AChE (Figs. 6C, D). By 16 days most endplates were clearly stained and showed the broad thick staining that is characteristic of normal adult endplates (Figs. 6E, F). We then attempted to correlate the appearance of focal accumulations of AChE with the molecular forms of the enzyme present in the ectopically innervated region of the muscle. Bon et al., (1979) described six forms of mammalian AChE by their sedimentation coefficients. We have adopted their nomenclature for the peaks of activity on sedimentation gradients since we find the same peaks. There are three globular (G) forms, G1 (4 S), Gz (6 S), and Gq (10 S) which, by analogy with forms found earlier in electric organs of Electrophorus (Bon et al., 1976), are thought to consist of oligomers of a single catalytic subunit (the subscript is the number of catalytic subunits); and three asymmetric (A) forms: A4 (9 S), A8 (13 S), and AI2 (16 S) that are thought to contain a long, collagen-like tail with one, two or three of the globular tetramers attached to form the head. Measurements of the areas of peaks corresponding to G1 (the catalytic monomer) and Arz (the endplatespecific form) in 10 experiments are combined in Fig. 7 and velocity sedimentation gradients from one experiment are shown in Fig. 8. No Al2 was detected at 3 days at which time the AChE sedimentation profiles of ectopically innervated regions were indistinguishable from those of endplate-free regions of denervated muscles lacking a foreign nerve. Between Days 3 and 5,
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ET AL.
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Rat Neuromuscular
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FIG. 5. Morphology of ectopic endplates. Longitudinal sections of ectopically innervated soleus muscles were stained for AChE and impregnated with silver to reveal nerve fibers. Ectopic endplates are shown 3 days (A), 5 days (B), 1 week (C), 2 weeks (D), and 1 month (E) after cutting the original nerve. A normal endplate is shown in (F). Bar = 25 /.rrn.
there was a large increase in G1, and a small amount of Alz appeared. There was a three- to four-fold increase in Alz between Days 5 and 7. Also, the amount of As doubled and the amount of G1 continued to increase. Over the next few weeks the amount of AI2 more than doubled again. G1 and A8 also increased although to a lesser extent, while the amount of G4 remained nearly constant throughout the period of new endplate formation. Thus, there is good agreement among the various methods we used to detect AChE. Between 3 and 5 days after cutting the original nerve, histochemically detectable AChE appears at sites that may be intracellular, and the level of G1, which is thought to be a pre-
cursor of the larger forms (Rieger et al., 1976; Vigny et al., 1976), rises. Focal accumulations of AChE are first detected at ectopic endplates 2-3 days later, by both histochemical and immunochemical methods, coincident with a large increase in the amount of Au+ Histochemical, immunochemical, and biochemical analysis all show further, gradual increases in AChE as the ectopic endplates mature. Synaptic
Basal Lam&a
A layer of basal lamina completely ensheaths each adult muscle fiber, extending through the synaptic cleft and into the postjunctional folds. Recent experiments
262
FIG. 6. Accumulation stained with anti-AChE and rhodamine (right) Bar = 20 Km.
DEVELOPMENTAL
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of AChE at ectopic endplates shown by immunofluorescence. Cross-sections of ectopieally innervated muscles were followed by fluorescein-second antibody and rhodamine-bungarotoxin. Each field is shown under fluorescein (left) optics. Ectopic endplates are shown 6 days (A, B), 8 days (C, D), and 16 days (E, F) after cutting the original nerve.
have shown that the synaptic portion of the basal lamina is specialized at adult NMJs (Sanes and Hall, 1979) and that during reinnervation of muscles (Sanes et CL, 1978) or regeneration of damaged muscles (Burden et al., 1979), the synaptic basal lamina plays a major role in guiding the differentiation of the pre- and postsynaptic membranes. To see if, and when, the basal lamina at ectopic synapses becomes specialized, we examined developing ectopic endplates with two antisera that recognize components of synaptic basal lamina (Sanes and Hall, 1979). One was made against a muscle basement membrane collagen fraction and then adsorbed with endplate-free regions of muscle; the other was made against bovine anterior lens capsule. Both sera contain antibodies to synaptic portions of adult muscle fibers’
basal lamina sheath; antilens capsule also stains blood vessels and the perineurial sheath surrounding nerves. These two sera recognize determinants in synaptic basal lamina that are distinct from each other and from AChE (Sanes and Hall, 1979). When newly formed ectopic endplates were examined 31 a.+Not
4
denewated
1 b.+Five
FIG. 7. Summary of changes in activity of G,, the catalytic subunit monomer of AChE, and Am the endplate form of AChE. Activities were determined by measuring the areas under peaks on velocity sedimentation gradients (see Fig. 8) of ectopic endplate-containing regions of soleus muscles. Activities are shown as a function of time after denervation; each point represents a single experiment.
~
t c. One week
Fraction
DAYS AFTER 0ENER”AwJN
days
number
FIG. 8. Molecular forms of AChE at ectopic endplates. The region of a soleus muscle containing newly formed ectopic endplates was homogenized, run on a sucrose density gradient, and the fractions were assayed for AChE activity. Sedimentation coefficients for peaks were as follows: Gr, 3.8-4.1 S; G,, 10.0-10.3 S; As, 12.6-13.1 S; A12 15.816.6 S. Peaks corresponding to G2 and Ad are small and poorly resolved under these conditions. Gradients are shown of ectopically innervated regions 0 days (A), 5 days (B), 1 week (C), and 3 weeks (D) after cutting the original nerve. Sedimentation runs from left to right.
263
FIG. 9. Accumulation of muscles were stained with Each field is shown under (E, F), and 30 days (G, H)
synaptic basal lamina at ectopic endplates shown by immunofluorescence. Cross-sections of ectopically innervated adsorbed antibasement membrane collagen followed by fluorescein-second antibody and rhodamine-bungarotoxin. fluorescein (left) and rhodamine (right) optics. Ectopic endplates are shown 3 days (A, B), 6 days (C, D), 8 days after cutting the original nerve. Bar = 20 pm.
with the adsorbed antibasement membrane collagen serum no binding could be detected for the first 4 days after denervation (Figs. 9A, B). At 6 days faint, thin lines of staining were found at some of the receptor clusters (Figs. 9C, D). Most of these endplates were still visible even when the fluorescein-second antibody had been diluted 1:8 with unconjugated-second antibody (see Methods); therefore, this staining demonstrates a substantial accumulation of the synapse-specific antigens between Days 4 and 6. About half of the clusters stained with this antiserum at 8 days, while other clusters in the same section were not detectably stained (Figs. 9E, F). By 2 weeks after denervation almost all of the ectopic endplates were stained by this antiserum, and at 1 month after denervation (Figs. 9G, H) virtually all the endplates appeared as thick bands similar to normal endplates. At some older ectopic synapses (Figs. 9G, H), as at normal adult endplates (Sanes and Hall, 1979), the staining with adsorbed antibasement membrane collagen serum extended slightly beyond the borders of the rhodamine-BuTx-stained patch of AChRs. The results of staining with the antilens capsule serum were generally similar to those with the anti-
basement membrane serum: some endplates were stained on Day 6, about half were stained on Day 8, and nearly all were stained by 2 weeks. The initially thin line of staining became thicker by 1 month; however, unlike the adsorbed antibasement membrane collagen serum, the staining with antilens capsule was always coextensive with the AChR clusters. There may have been some staining 4 days after denervation since endplates were always among the brightest regions of the fiber surface; however, since the entire surface of soleus muscle fibers, unlike diaphragm muscle fibers (Sanes and Hall, 1979), was lightly stained by antilens capsule, it was difficult to determine if endplates were specifically stained at 4 days. In summary, the basal lamina of adult muscle fibers becomes specialized during the formation of new synaptic sites, Synapse-specific antigens are first detectable at some ectopic synapses by 6 days after denervation, and they accumulate gradually over the next few weeks until all the endplates appear mature. We have also found that cutting the foreign nerve on Day 4 prevents the accumulation of the synaptic basal lamina antigens at ectopic endplates (data not shown).
DEVELOPMENTALBIOLOGY
264 DISCUSSION
We have studied the formation and maturation of new, ectopic, neuromuscular synapses between an implanted foreign nerve and soleus muscle fibers in adult rats. We focused on the differentiation of four molecular components of the synapse: AChRs which are integral membrane proteins concentrated at the crests of the subsynaptic folds; AChE which is concentrated at the synapse, and at least some of which is associated with synaptic basal lamina; and two immunologically defined components of the synaptic basal lamina. We find that all of these components accumulate at ectopic synapses in less than a month after the original nerve is cut. AChRs cluster before specialized basal lamina components including AChE accumulate in detectable amounts. Clustered AChRs become densely packed and metabolically stable before AChE accumulates. Synapse-specific components of the basal lamina accumulate concurrently with or just before accumulation of AChE. The first detectable event occurs 2 days after the original nerve is cut, when clusters of AChRs appear underneath the ectopic nerve. In electrophysiological studies of ectopic synapse formation on rat soleus muscles, Ldmo and Slater (1976, 1978) found that the earliest signs of transmission occurred at 2 l/2-3 days. Receptors in the first clusters to be detected have a moderately high density, about half of their final density. The final density is achieved within a few days after the clusters form. This change may correspond to the transition from “aggregates” to “plaques” of AChRs recently described by Steinbach (1981) and the development of resistance to dispersal by low Ca2+ recently described by Bloch and Steinbach (1981) during synapse formation in developing muscle. The rate of degradation of clustered AChRs in rat muscle decreases 5- to lo-fold within the first few days after clustering at both ectopic and embryonic NMJs (Reiness and Weinberg, 1981). The accumulation of AChE was studied immunocytochemically, histochemically, and by sedimentation analysis. All three methods first detected a high concentration of synaptic AChE at the ectopic endplates around 1 week after cutting the original nerve, and the amount continued to increase over the next 2 weeks. Two components of the NMJ, that in adult muscle are associated with the basal lamina (Sanes and Hall, 1979), also occur at mature ectopic NMJs. Synapse-specific antigens were detected at some endplates as early as Day 6; the fraction of endplates containing these antigens increased during the next week until by 2 weeks they were present at virtually all endplates. Because
VOLUME 84, 1981
ectopic NMJs form around a preexisting basal lamina that is initially extrasynaptic, a process of remodeling must occur in the extracellular matrix (as well as in the muscle’s plasma membrane) as the synaptic matures. At synapses formed during normal development (Kelly and Zacks, 1969; Jacob and Lentz, 1979) and in cell culture (Frank and Fischbach, 1979), the basal lamina appears between the nerve terminal and the muscle fiber at a very early stage. Whether this structure contains the synaptic antigens from the beginning of whether some form of remodeling of the basal lamina takes place during development is not yet known. The earliest times at which AChR clusters (Bevan and Steinbach; 1977; Braithwaite and Harris, 1979), accumulation of AChE at synaptic sites (Bennett and Pettigrew, 1974), A1z, the endplate form of AChE (Vigny et al., 1976), and basal lamina in the synaptic cleft (Kelly and Zacks, 1969) have been reported in rat embryos are all around the 15th or 16th days of gestation. Thus, the sequence of steps in synapse formation is difficult to resolve in the embryo, although it seems likely that AChE accumulates after receptors cluster (Rubin et al., 1979; Kullberg et al., 1980). Our observations at newly formed ectopic NMJs clearly separate the clustering of AChRs from the focal accumulation of AChE at synapses. Furthermore, in contrast to the results in rat embryos Reiness and Weinberg, (see 1981), AChR density and metabolic turnover time reached nearly mature values at ectopic synapses before detectable amounts of AChE had accumulated. Our results also suggest that the remodeling of the basal lamina when new synapses are formed on adult muscle fibers is concurrent with or slightly precedes the accumulation of AChE, which is associated with the basal lamina at adult NMJs (Hall and Kelly, 1971; Betz and Sakmann, 1974; McMahan et al., 1978). The mechanisms by which the postsynaptic membrane and the synaptic basal lamina are assembled are unknown; however, our results show that they are not formed as a unit. Recent experiments have shown that components of the synaptic basal lamina play important roles in the differentiation of regenerating axons into nerve terminals when the original synaptic sites are reinnervated (Sanes et al., 1979) and in the clustering of AChRs when damaged muscle regenerates (Burden et aZ., 1979). It is an attractive possibility that during the formation of new NMJs, components of the basal lamina may play a role in directing the specialization of other components of the synapse. Our experiments clearly show that complete differentiation of the basal lamina at newly forming ectopic synapses takes place well after functional transmission is established and the initial clustering of receptors has occurred. However, our exper-
WEINBERG ET AL.
Adult Rat Neuromuscular
iments provide no evidence on whether unknown antigens or low levels of known antigens play a role in the formation of the NMJ, or whether components of the synaptic basal lamina regulate later steps in the maturation of pre- and postsynaptic membranes. We thank Miu Lam for expert technical assistance, and Eric Frank, who first demonstrated the procedure for forming ectopic endplates to us. This work was supported by grants from the Muscular Dystrophy Association (MDA), the National Institutes of Health (NIH), and the National Science Foundation to Z.W.H. C.B.W. was supported by an NIH training grant and J.R.S. by a postdoctoral fellowship from MDA REFERENCES AITKEN, J. T. (1950). Growth of nerve implants in voluntary muscle. J. Anat. 34, 38-49. BEERMAN, D. H., and CASSENS, R. G. (1976). A combined silver and acetylcholinesterase method for staining intramuscular innervation. Stain Technol. 51.173-177. BENNETT, M. R., and PETTIGREW, A. G. (1974). The formation of synapses in striated muscle during development. J. Physiol. 241, 515545. BENNETT, M. R., and PETTIGREW, A. G. (1975). The formation of neuromuscular synapses. Cold Spring Harbor Symp. Quant. BioL 49, 409-424. BET& W., and SAKMANN, B. (1973). Effects of proteolytic enzymes on function and structure of frog neuromuscular junctions. J. Physiol. 230,673-688. BEVAN, S., and STEINBACH, J. H. (1977). The distribution of a-bungarotoxin binding sites on mammalian skeletal muscle developing in vivo. J. Physiol. 267, 195-213. BLOCH, R. J., and STEINBACH, J. H. (1981). Reversible disruption of acetylcholine receptor clusters at the developing rat neuromuscular junction. Develop. BioL 81, 386. BON, S., HUET, M., LEMONNIER, M., RIEGER, F., and MASSOULIE, J. (1976). Molecular forms of Electrophorus acetylcholinesterase. Eur. J. Biochem. 68,523-530. BON, S., VIGNY, M., and MASSOULIE, J. (1979). Asymmetric and globular forms of acetylcholinesterase in mammals and birds. Proc. Nat. Acad Sci. USA 76,2546-2550. BRAITHWAITE, A. W., and HARRIS, A. J. (1979). Neural influence on acetylcholine receptor during embryonic development of skeletal muscles. Nature (London) 279, 549-551. BURDEN, S. (1977). Development of the neuromuscular junction in the chick embryo: the number, distribution, and stability of acetylcholine receptors. Develop. Biol. 57,317-329. BURDEN, S. J., SARGENT, P. B., and MCMAHAN, U. J. (1979). Acetylcholine receptors in regenerating muscle accumulate at original synaptic sites in the absence of the nerve. J. Cell BioL 82,412-425. EDWARDS, C. (1979). The effects of innervation on the properties of acetylcholine receptors. Neuroscience 4, 565-584. ELMAN, G. L., COURTNEY, K. D., ANDRES, V., and FEATHERSTONE, R. M. (1961). A new and rapid calorimetric determination of acetylcholinesterase activity. Biochem. Pharmacology 7, 88-95. ELSBERG, C. A. (1917). Experiments on motor nerve regeneration and the direct neurotization of paralyzed muscles by their own and foreign nerves. Science 45, 318-320. FEX, S., SONNESSON,B. THESLEFF, S., and ZELENA, J. (1966). Nerve implants in botulinum poisoned mammalian muscle. J. Phusiol. 184, 872-882.
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FEX, S., and THESLEFF, S. (1967). The time required for innervation of denervated muscles by nerve implants. Life Sci. 6,635-639. FRANK, E., and FISCHBACH, G. D. (1979). Early events in neuromuscular junction formation in vitro. J. Cell Biol. 83, 143-158. FRANK, E., GAUTVIK, K., and SOMMERCHILD, H. (1975). Cholinergic receptors at denervated mammalian motor end-plates. Acta. PhpsioL Stand. 95, 66-76. GREENBERG, A. J., PARKER, K. K., and TREVOR, A. J. (1977). Immunochemical studies of mammalian brain acetylcholinesterase. J. Neurochem. 29,911-917. GUTH, L., and ZALEWSKI, A. A. (1963). Disposition of cholinesterase following implantation of nerve into innervated and denervated muscle. Exp. Neurol. 7.316-326. GUTMANN, E., and HANZLIKOVA, V. (1967). Effects of accessory nerve supply to muscle achieved by implantation into muscle during regeneration of its nerve. Physiologia Bohemoslovaca 16,244-250. GIJTMANN, E., and YOUNG, J. Z. (1944). The re-innervation of muscle after various periods of atrophy. J. Anat. 78.15-43. GWYN, D. G., and AITKEN, J. T. (1964). New motor end-plates and their relationship to muscle fibre injury. Nature (London) 203,651652. GWYN, D. G., and AITKEN, J. T. (1966). The formation of new motor endplates in mammalian skeletal muscle. J Anat. 100, 111-126. HALL, Z. W. (1973). Multiple forms of acetylcholinesterase and their distribution in endplate and non-endplate regions of rat diaphragm muscle. J. Neurobiol. 4, 343-361. HALL, Z. W., and KELLY, R. B. (1971). Enzymatic detachment of endplate acetylcholinesterase from muscle. Nature New BioL 232,6263. JACOB, M., and LENTZ, T. L. (1979). Localization of acetylcholine receptors by means of horseradish peroxidase-a-bungarotoxin during formation and development of the neuromuscular junction in the chick embryo. J. Cell Biol. 82, 195-211. JANSEN, J. K. S., LIMO, T., NICOLAYSEN, K., and WESTGAARD, R. H. (1973). Hyperinnervation of skeletal muscle fibers: Dependence on muscle activity. Science 181, 559-561. KELLY, A. M., and ZACKS, S. I. (1969). The fine structure of motor endplate morphogenesis. J. Cell BioL 42.154-169. Ko, P. K., ANDERSON, M. J., and COHEN, M. W. (1977). Denervated skeletal muscle fibers develop discrete patches of high acetylcholine receptor density. Science 196.540-542. KOENIG, J. (1971). Contribution a l’etude de la neoformation experimentale des plaques matrices de rat. Arch. Anat. Microscop. MOTphol. Exp. 60, l-26. KORNELIUSSEN, H., and SOMMERSCHILD, H. (1976). Ultrastructure of the new neuromuscular junctions formed during reinnervation of rat soleus muscle by a “foreign” nerve. Cell Tiss. Res. 167.439-452. KUFFLER, D., THOMPSON, W., and JANSEN, J. K. S. (1977). The elimination of synapses in multiply innervated skeletal muscle fibres of the rat: Dependence on distance between endplates. Brain Res. 138.353-358. KULLBERG, R. W., MIKELBERG, F. S., and COHEN, M. W. (1980). Contribution of cholinesterase to developmental decreases in the time course of synaptic potentials at amphibian neuromuscular junctions. Develop. Biol. 75, 255-267. LENTZ, T. L. (1969). Development of neuromuscular junctions in regenerating limbs of the newt Triturus. J. Cell Biol. 42.431-443. LIMO, T., and SLATER, C. R. (1976). Control of neuromuscular synapse formation. In “Synaptogenesis” (L. Taut, ed.), pp. 9-30. Naturalia & Biologia, Jouy en Josas. LIMO, T., and SLATER, C. R. (1978). Control of acetylcholine sensitivity and synapse formation by muscle activity. J. PhysioL 275,391-402. MCMAHAN, U. J., SANES, J. R., and MARSHALL, L. M. (1978). Cholin-
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esterase is associated with the basal lamina at the neuromuscular junction. Nature (London) 271, 172-174. MILEDI, R. (1963). Formation of extra nerve-muscle junctions in innervated muscle. Nature (London) 199.1191-1192. REINESS, C. G. and WEINBERG, C. B. (1981). Metabolic stabilization of aeetylcholine receptors at newly formed neuromuscular junctions in rat. Develop. Biol. 83, 247-254. RIEGER, F., FAIVRE-BAUMAN, A., BENDA, P., and VIGNY, M. (1976). Molecular forms of acetylcholinesterase: Their de nowo synthesis in mouse neuroblastoma cells. J. Neurochem. 27,1059-1063. RUBIN, L. L., SCHUETZE, S. M., and FISCHBACH, G. D. (19’79). Aeeumulation of acetylcholinesterase at newly formed nerve-muscle synapses. Develop. Biol. 69, 46-58. SANE&J. R., and HALL, 2. W. (1979). Antibodies that bind specifically to synaptic sites on muscle fiber basal lamina. J. Cell Biol. 83, 357370. SANES, J. R., MARSHALL, L. M., and MCMAHAN, U. J. (1978). Reinnervation of muscle fiber basal lamina after removal of myofibers. Differentiation of regenerating axons at original synaptic sites. J. Cell Biol. 78, 176-198.
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STEINBACH, J. H. (1981). Developmental changes in acetylcholine receptor aggregates at rat skeletal muscle neuromuscular junctions. Develop. Biol 84, 267-276. VIGNY, M., KOENIG, J., and RIEGER, F. (1976). The motor end-plate specific form of acetylcholinesterase: Appearance during embryogenesis and re-innervation of rat muscle. J. Neurochem. 27,13471353. WAERHAUG, O., KORNELIUSSEN, H., and SOMMERSCHILD, H. (1977). Morphology of motor nerve terminals on rat soleus muscle fibers reinnervated by the original and by a “foreign” nerve. Anat. Embrgol. 151, 1-15. WAKE, K. (1976). Formation of myoneural and myotendinous junctions in the chick embryo. Cell Tiss. Res. 173,383-400. WEINBERG, C. B., and HALL, Z. W. (1979a). Junctional form of acetylcholinesterase restored at nerve-free endplates. Develop. Biol. 68, 631-635. WEINBERG, C. B., and HALL, Z. W. (197913). Antibodies from patients with myasthenia gravis recognize determinants unique to extrajunctional acetylcholine receptors. Proc. Nat. Acad. Sci. USA 76, 504-508.