DEVELOPMENTAL BIOLOGY 81,386-391 (1981)

Reversible Loss of Acetylcholine Receptor Clusters at the Developing Rat Neuromuscular Junction

ROBERT J. BLOCH AND JOE HENRY STEINBACH

The Neurobiology Laboratory, The Salk Institute, P.O. Box 85800, San Diego, California 92138

Received April 18, 1980; accepted in revised form July 28, 1980

Exposure of sternomastoid muscles excised from Is-day embryonic rats to medium depleted of Ca” or containing high concentrations of KC1 leads to extensive loss of aggregates of acetylcholine receptors newly formed at the motor end plate region. Upon restoration of Ca2+ or removal of excess KCl, receptor accumulations reappear in the central regions of about one-third of the muscle fibers. This susceptibility of junctional AChR aggregates lasts only a short while during development of the neuromuscular junction. By the time of birth, end plate receptor aggregates have become resistant to these treatments.

INTRODUCTION

During neuromuscular junction formation, one of the earliest morphological changes observed at the post- synaptic membrane is the aggregation there of acetyl- choline receptors (AChRs; Bevan and Steinbach, 197’7; Burden, 1977; Jacob and Lentz, 1979). In the sterno- mastoid muscle of the rat, these aggregates first appear on Days 15 to 16 of embryogenesis (Bevan and Stein- bath, 1977), and coincide with regions of acetylcholin- esterase and nerve staining (Bevan and Steinbach, 1977; Harris, submitted for publication). We therefore con- sider them to occur at the developing motor end plate.

AChRs also cluster in the plasma membranes of rat myotubes maintained in tissue culture (Axelrod et al., 1976; Bloch, 1979). These clusters can be reversibly dis- persed by several treatments: by inhibitors of energy metabolism, carbachol, Ca2+-depleted medium or high concentrations of KC1 (Bloch, 1979, and unpublished). We have examined the susceptibility of embryonic and neonatal end plate AChR aggregates to the same treat- ments. Our observations show that, like the AChR clus- ters of cultured myotubes, the earliest junctional re- ceptor aggregates are unstable assemblages, and suggest that the mechanism which stabilizes junctional recep- tor aggregates may change during embryogenesis.

METHODS

In most of the experiments reported here, we used the following basic protocol. Sternomastoid muscles from embryonic or neonatal Sprague-Dawley rats were dissected and pinned to sterile Sylgard (Dow-Corning, Midland, Mich.) coated petri dishes. They were bathed in Dulbecco-Vogt modified Eagle’s medium (DMEM) containing 1% fetal calf serum (Associated Biomedic

Systems, Buffalo, N.Y.) and buffered to pH 7.0 with N-2-hydroxyethylpiperazine-w-2-ethanesulfonic acid (Hepes). Muscles were first stained for 1 hr at 22°C in an atmosphere of 100% O2 with 5 pg/ml monotetra- methylrhodamine-a-bungarotoxin (R-aBT), prepared as reported (Anderson and Cohen, 1974; Ravdin and Axelrod, 1977), in Hepes-buffered DMEM plus 5% fetal calf serum. After two washes to remove excess unbound toxin, muscles received 10 ml of DMEM plus 5% fetal calf serum or medium modified according to the exper- iment. Muscles were then incubated for 6 hr at 37°C in an atmosphere of 95% 02, 5% C02, as described (Merlie et al., 1979), after which they were washed, fixed in 4% paraformaldehyde in 0.1 M Na phosphate buffer, pH 7.2, glycerinated, teased, and mounted for fluorescence observations. Fluorescence microscopy and photo- graphic methods have been described (Bloch, 1979).

In some experiments, we varied our basic protocol slightly. To determine if the loss of end plate AChR clusters was due to dissociation of R-aBT label, we stained the muscles after but not before treatment, or we stained them both before and after treatment. As discussed below, we observed no differences in the re- sults using these altered protocols, except in the case of cluster loss caused by carbachol. We employed a sec- ond variation of this protocol to determine the revers- ibility of cluster loss. For this experiment, we followed the basic protocol for the initial labeling and 6-hr in- cubation, but instead of fixing the muscles at the end of this period we placed them into control medium and incubated them for an additional 16 hr. Most muscles were then fixed and restained with R-CYBT, and mounted and observed under fluorescence illumination.

Muscles were treated in several ways to test the sus- ceptibility of the AChR aggregates at the motor end

386 0012-1606/81/020386-06$02.00/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

BRIEF NOTES 387

plate to disruption. One method was to expose the mus- cles for 6 hr to medium depleted of Ca’+. Ca2+ depletion was achieved either by (i) using DMEM prepared with- out CaClz and supplemented with dialyzed fetal calf serum, or (ii) bringing DMEM plus 5% fetal calf serum to 10 mM EGTA and adding CaC& to adjust the cal- culated final free Ca2+ concentration to 0.1 mM, or to 1.8 mM for controls (calculation done assuming an af- finity of EGTA for Ca2+ of 2 X 10m6 M; see Ogawa, 1968). This control value was chosen because 1.8 mM is the Ca2+ concentration of unmodified DMEM. The two methods gave similar but not identical results (see Re- sults).

We also exposed muscle fibers to high concentrations of K’ for 6 hr. For these experiments, a modified DMEM was prepared in which NaHC03 and NaCl were replaced with KHC03 and either KC1 or K methane sulfonate (Eastman). To minimize damage caused by strenuous contraction of muscles exposed to these solutions, the K+ concentration of the medium bathing the muscles was raised gradually over a period of several minutes, until the final concentration (150 mM) was achieved. The normal concentration of K+ in DMEM is 5.4 mM.

A third method we used to induce the loss of end plate AChR aggregates was continuous exposure to car- bachol, a receptor agonist, at a concentration of 0.1 mM.

RESULTS AND DISCUSSION

Most muscle fibers of sternomastoid muscles removed from 16-day embryonic rats and stained with R-aBT displayed AChR aggregates. Such aggregates, usually found approximately in the middle of the fiber lengths, are 22 * 6 pm in length (means + SD) and consist of many smaller speckles (Steinbach, in press). A signif- icant number (15-30%) of muscle cells did not have detectable AChR aggregates (Table 1). These cells, which were not characterized further, may have been myotubes or myofibers which had not yet received mo- tor innervation. The AChR aggregates present in 16- day embryonic sternomastoid muscles were stable dur- ing prolonged incubation in control culture medium (Fig. 1A). But AChR aggregates were lost when muscles were incubated in media containing lowered concentra- tions of Ca2+ or elevated concentrations of KC1 (Figs. lC, E), conditions which disrupt the AChR clusters of cultured rat myotubes (Bloch, 1979, and unpublished).

These observations were quantitated by counting the number of muscle fibers in teased preparations which displayed control receptor clusters. Control receptor clusters had intense fluorescent R-(uBT labeling showing clear boundaries with the surrounding, less intensely stained plasma membrane. AChR aggregates were con- sidered to be absent from those end plate regions which

TABLE 1 PERCENTAGE OF MYOFIBERS WITH AChR AGGREGATES FOLLLOWING

VARIOUS EXPERIMENTAL TREATMENTS

Treatment”

I. After 6 hr incubation in

indicated medium

II. After 16 hr additional

incubation in control medium

Controls 1. Unmodified 2. Buffered to 1.8 mM

free Ca2+ with EGTA

Low Ca’+ 3. CaClz omitted 4. Buffered to 0.1 mM

free Ca+ with EGTA

High K+ (150 mhf) 5. KC1 + KHC03 6. K-methane-

sulfonate + KHC03

Carbachol (0.1 mM) 7. Without R-oBT

prestain 8. With R-aBT

prestain Other

9. Sodium azide (5 mlK)

10. Damagedb

78 + 3 (6) 77,86

75 f 5 (3) -C

4 f 3 (3) 2837

23 f 10 (3) -

20 f 7 (3) 45,58

85,89 -

30,33 -

70,86 -

81 ? 5 (3) - 100,79 -

Note. Sternomastoid muscles from 16-day embryonic rats were treated as described in Fig. 1 and Methods. Values given are the mean percentage of muscle fibers which showed “control” aggregates, + 1 SD, with the number of experiments in parentheses. When only two experiments were quantitated, the results of individual experiments are given. Results given in Column I were obtained immediately after 6 hr of culture under the indicated conditions; staining of these mus- cles with R-(YBT was performed at the start of the experiment, unless otherwise indicated. Some muscles were allowed to recover in control medium for 16 hr after a 6-hr period under the indicated conditions (Column II); staining of these muscles with R-aBT was at the start of the experiment and again after the 16-hr recovery period. At least 50 muscle fibers were examined for each value presented.

a All media used were DMEM, or DMEM modified as indicated, and contained 5% (v/v) fetal calf serum; in Case 3, and in some of the samples of Case 1, the fetal calf serum was dialyzed against phos- phate-buffered saline before use. DMEM, otherwise unchanged, con- tained 40 mM KHCOI and either 110 mM KC1 or 110 mM K-meth- anesulfonate, instead of NaHC03 and NaCl, plus 4 m&f NaCl.

b Damaged by breaking myofibers near both tendon insertions using forceps.

‘Not determined.

displayed much less intense fluorescent label, which were less than 5 pm in length, and lacked clear borders with surrounding membrane.

Using these criteria for quantitation, we found that 60 to 80% of the AChR aggregates in the end plate regions of 16-day embryonic sternomastoid muscles

388 DEVELOPMENTAL BIOLOGY VOLUME 81, 1981

FIG. 1. Loss and reappearance of AChR clusters of 16-day embryonic rat sternomastoid muscles. The sternomastoid muscles of Is-day embryonic rats were stained with R-(YBT and then exposed to different experimental conditions for 6 hr, as described in the basic protocol in Methods. (A, B) Control medium; (C, D) high KC1 medium; (E, F, H) medium prepared free of CaClz and containing dialyzed fetal calf serum; (G) medium containing 5 mM sodium azide. After 6 hr treatment, some samples were removed from incubation and fixed in para- formaldehyde (A, C, E, G). Others were washed and incubated further in control medium for 16 hr (B, D, F, H), and then fixed. Samples B, D and F were restained with R-(uBT after fixation; sample H was not restained, so all fluorescence observed in H is probably due to R-aBT bound to the muscle at the start of the experiment, 22 hr earlier. The arrows in C and E point to speckles of R-(YBT stain which may be the remnants of disrupted end plate aggregates. The arrowhead in G indicates one of the many AChR aggregates in the field which remain intact after exposure to sodium azide. The arrow in F points to an end plate aggregate, slightly out of focus, seen face on; the arrowhead points to an aggregate seen edge on. The brightly staining masses in the upper parts of B and F are autofluorescent debris. The higher background seen in F is due to the shorter times used for printing this image. The scale bar in H represents 50 pm.

were lost during continuous exposure to high concen- Ca2+ concentrations which were low but uncontrolled, trations of KC1 or to low concentrations of Cazi (Table or by buffering with EGTA to give a free Ca2+ concen- 1). The decreased free Ca2+ concentrations were achieved tration of 0.1 m&L These two methods gave similar but by using medium prepared without CaC12, affording not identical results (Table 1). This suggests both that

BRIEF NOTES 389

FIG. 2. ‘AChR aggregates of neonatal sternomastoid muscles are resistant to disruption. The procedure described in Fig. 1 was followed for the initial 6-hr treatment, but muscles from newborn, less than 24-hr-old (a-c) and from 18-day-old (d-f) rats, were used. End plates observed en face were photographed; representative examples are presented here. (a, d) Control medium; (b, e) medium buffered to 0.1 mMfree Ca2+ using EGTA (see Methods); (e, f) high KC1 medium. No effect of either low Ca2+ medium or high KC1 medium was observed in two experiments. The scale bar represents 25 P.

the effect of EGTA is due primarily to its Ca2+ chelating activity, and that removing CaC12 from the medium can effectively lower free Ca2+ concentrations nearly as well as EGTA; indeed, the greater loss of AChR aggregates in medium prepared without CaC12 was probably due to the presence of free Ca2+ concentrations considerably lower than 0.1 mM.

Treatment of 16-day rat sternomastoid muscles for 6 hr with carbachol also caused a marked loss of end plate AChR aggregates, and this loss was blocked by preincubation of the muscles with R-(uBT (Table 1). This was the only instance in which prestaining with R-cuBT affected the experimental results. In experiments using low Ca’+ or high KC1 concentrations, similar results were obtained when muscles were stained either before or after treatment (data not shown).

The above results show that, at early stages of neu- romuscular junction formation, the postsynaptic AChR aggregates are susceptible to dispersal or loss. To learn how long after onset of synapse formation this suscep- tibility lasts, we examined muscles taken from neonatal rats. We found that in muscle from newborns (~24 hr old) the end plate receptor aggregates were much more resistant (>‘70% fibers with clusters, analyzed as for embryonic muscles) to low Ca2+ or high KC1 treatments (Figs. 2a-c) or to carbachol (not shown). By 18 days postnatal, when junctional morphology resembles that at adult neuromuscular junctions (Steinbach, in press), no effect of low Ca2+ medium and only marginal blur- ring of aggregates in high Kf medium were found, in-

dicating that end plate receptor clusters were by this time nearly completely resistant to these treatments (Figs. 2d-f).

We do not understand the mechanism by which these treatments cause the loss of AChR aggregates on em- bryonic muscle fibers. Loss is probably not due to dis- sociation of the fluorescent a-BT label, as muscles stained with R-aBT after treatment gave results iden- tical to those stained before, when dispersal was pro- duced by low Ca2+ or high KC1 (not shown). The car- bachol effect may be mediated through activation of the AChR, since in this case prestaining with R-(YBT blocked dispersal. Neither membrane depolarization nor decreased concentrations of Na+ appear to be the cause of cluster dispersal induced by high KC1 concen- trations. When we exposed muscles to high K+ medium in which chloride was replaced with methane sulfonate, end plate receptor aggregates, despite the presence of 150 mM K+, were not lost (Table 1). This observation suggests that other factors, perhaps osmotic swelling and ionic changes accompanying swelling, are respon- sible for cluster loss in high KC1 medium. Swelling alone does not appear to be sufficient, however. In two experiments we found that incubation in hypotonic medium (six parts DMEM plus 5% fetal calf serum and four parts of water) following the basic protocol, caused the loss of only 5% of end plate receptor clusters. It is possible that cluster loss, in general, can be caused by alterations in intracellular ionic concentrations.

We do not think it likely that cluster loss was due to damage incurred by the muscles, for the following rea- sons. (i) Cutting some of the muscle fibers either near their insertion points or close to the end plates did not cause AChR clusters to be lost (Table 1). (ii) The rate of protein synthesis by muscles in Ca2+-depleted me- dium was the same as in controls. In one experiment, [3H]leucine (10 &i/ml) was introduced into the medium prepared free of Ca2+ during the last 2 hr of incubation. After extensive washing, muscles were removed from their insertions and dissolved overnight in 0.1 N NaOH. TCA-precipitable counts per milligram protein (mea- sured by the method of Lowry et al., 1951) were found to be identical (1.2 x lo3 cpm/mg protein) between mus- cles incubated in control DMEM and low Ca2+ medium. (iii) Treatment with 5 mM azide in control medium fol- lowing the basic protocol did not result in cluster loss (Table 1, Fig. 1G). However, treatment with 5 mM azide plus 25 mM 2-deoxyglucose did cause loss of the AChR aggregates, and also caused extensive fiber damage as evidenced by swollen, broken fibers and by an increase in autofluorescence of the fibers (not shown). Such ev- idence of fiber damage was not seen in muscles exposed to carbachol or to high KC1 or low Ca2+ concentrations.

An important observation is that the dispersal of the

390 DEVELOPMENTAL BIOLOGY VOLUME 81, 1981

AChR clusters by these treatments was at least par- tially reversible. Matched pairs of muscles from the same 16-day embryonic litter were treated in medium prepared free of CaClz for 6 hr following the basic pro- tocol. One muscle from each pair was then removed from incubation and fixed; subsequent observation showed less than 10% of its fibers to have AChR ag- gregates. The other muscle from each pair was placed into control medium and allowed to incubate for an additional 16 hr. After restaining, nearly five times more of the fibers displayed AChR aggregates. Simi- larly, one of two paired muscles exposed to high KC1 medium showed more than a twofold recovery of AChR aggregates when incubated for 16 hr in control medium. Upon recovery from both treatments, aggregates ap- peared approximately in the middle of the lengths of the fibers and, when present on adjacent fibers, seemed to be aligned (Figs. lD, F, H). Data obtained from two such experiments are presented in Table 1. Reversibility of the carbachol effect was not examined.

The development of junctional AChR aggregates re- sistant to low Ca2+ or high KC1 treatments occurs within several days after innervation begins. By the time of birth (within 6 days of initial receptor cluster- ing), the postsynaptic receptor aggregates are nearly completely resistant to these treatments. The trans- formation from susceptibility to resistance occurs over the same time period as the appearance of metabolically stable junctional AChR (Berg and Hall, 19’75; Steinbach et al., 19’79; Reiness and Hall, 1979). This set of changes may define two stages in the early development of the motor end plate. The first stage, reached soon after in- nervation, is characterized by receptor accumulations which can be lost and in which AChRs are rapidly me- tabolized. It is probably formed by aggregation of ex- trajunctional AChRs under the nerve (Anderson and Cohen, 1977; Anderson et al., 1977; Cohen and Pumplin, 1979; Frank and Fischbach, 1979). The second stage, reached by the time of birth, has receptor aggregates which are both physically and metabolically stable.

An important question is whether the developmental change in susceptibility of AChR clusters that we ob- served is due to a change in the stability of the clusters themselves or, rather, to a change in the metabolism of the muscle fibers. We cannot answer this question at present, nor can we explain how these treatments cause the loss of receptor aggregates. Despite these difficulties in interpretation, we believe that the ability of embryonic end plate receptor clusters to be lost and to reform, and the loss of this ability upon maturation, may be useful in further experiments on the mechanism of neuromuscular junction formation.

Our results also suggest that further work on recep- tor clustering in cultured rat myotubes may be helpful

in understanding early junctional development. Al- though AChR cluster formed at the neuromuscular junction do not disperse in response to azide treatment, as found for myotube clusters in vitro (Bloch, 1979), clusters formed both in vivo and in vitro can be dis- persed by treatment with carbachol, or with low Ca2+ or high KC1 concentrations (Bloch, 1979, and in prep- aration). Together with the morphological similarities between junctional clusters on 16-day embryonic fibers and on cultured myotubes, these resemblances indicate that cluster formation in cultured rat myotubes and at developing junctions may involve similar mechanisms.

R.J.B. thanks Stephen Heinemann for his encouragement and fi- nancial support. Dr. Charles Edwards suggested the use of K-meth- anesulfonate to distinguish between osmotic and depolarizing effects of KCl. This work was funded by grants to J.H.S. from the National Science Foundation and the Muscular Dystrophy Association, and to Stephen Heinemann from the National Institutes of Health (2 ROI NS 11549) and from the Muscular Dystrophy Association. R.J.B. is the recipient of a senior research fellowship from the San Diego chap- ter of the American Heart Association, and the beneficiary of funds made available from a grant from the Samuel Roberts Noble Foun- dation of Ardmore, Oklahoma. J.H.S. is a Sloan Foundation Fellow.

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BRIEF NOTES 391

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