Acetylcholine receptors at the neuromuscular junction: Developmental change in receptor turnover

DEVELOPMENTAL

BIOLOGY

61, ‘79-85 (1977)

Acetylcholine Receptors at the Neuromuscular Junction: Developmental Change in Receptor Turnover STEVE BURDEN’ Department

of Pharmacology, and Department

Harvard Medical School, 25 Shattuck Street, Boston, Massachusetts of Zoology, University of Wisconsin, Madison, Wisconsin 53706

Received May 26,1977;

02115,

accepted in revised form June 23,1977

The turnover of acetylcholine receptors labeled with ““I-labeled a-bungarotoxin was measured in the developing posterior latissimus dorsi muscle of the chick. The degradation rates for acetylcholine receptors at the neuromuscular junction and in extrajunctional regions of the muscle cell were determined. One week after hatching, the rates of junctional and extrajunctional receptor degradation are identical (t,,, = 30 hr). Three weeks after hatching, however, the rate of junctional receptor degradation is considerably slower (t,,, 2 5 days) and different than the rate of extrajunctional receptor degradation (t,,, = 30 hr). Thus, receptors which are localized at the neuromuscular junction early in embryonic life only become stable several weeks after hatching. INTRODUCTION

muscle (Lemo and Rosenthal, 1972; Hogan et al., 1976) have been examined. Muscle activity appears to play an important role in this regulation. Our knowledge of the regulation of junctional AChRs is less complete. In this study, the regulation of junctional AChRs during development is investigated. Junctional and extrajunctional AChRs in denervated rat diaphragm muscle have different degradation rates (t; extrajunctional: ca. 20 hr; t; junctional: 2 5 days) (Berg and Hall, 1974, 1975; Chang and Huang, 1975). This observation is consistent with a mechanism whereby receptors would accumulate at the neuromuscular junction in response to a decrease in the rate of receptor degradation at the synapse. In embryonic chick muscle, however, the rates of junctional and extrajunctional receptor degradation are identical (t1,2 = 30 hr) (Burden, 1977al. Thus, receptors can accumulate at synapses without a change in their rate of degradation. It was concluded that the accumulation of receptors at these embryonic junctions is regulated either by preferential incorporation of re-

Acetylcholine receptors (AChRs) in innervated skeletal muscles of all adult vertebrates are highly localized to the neuromuscular junction. In addition to these junctional AChRs, embryonic muscle and denervated adult muscle possess large number of AChRs at extrajunctional regions of the muscle fiber. It is not clear how the distribution of these receptors is established and what factors regulate their maintenance. During development, high concentrations of AChRs are established at newly formed synapses (Anderson et al., 1976; Frank and Fischbach, 1977), and thereafter the extrajunctional receptor density decreases until the adult distribution is attained (Diamond and Miledi, 1962; Dennis and Ort, 1977; Bevan and Steinbach, 1977; Burden, 1977a). Some factors which regulate the number of extrajunctional receptors during embryonic development (Burden, 1977a; see also Gordon and Vrbova, 1975) and in denervated ’ Send reprint requests to author’s present address: Department of Neurobiology, Stanford University Medical School, Stanford, Calif. 94305.

79 Copyright All rights

0 197’7 by Academic Press, Inc. of reproduction in any form reserved.

ISSN

0012-1606

80

DEVELOPMENTAL BIOLOGY

ceptors at the synapse or by a restriction of receptor diffusion in the postjunctional membrane. In this study, the degradation rate of junctional AChRs has been measured in chicks after hatching, and the development of slowly degrading junctional receptors has been measured. MATERIALS

AND

METHODS

Experiments were performed on White Leghorn chicks (hatchlings to 18 weeks posthatch) obtained from SPAFAS. Purification and iodination of a-bungarotoxin and autoradiographic techniques have been described (Burden, 1977a,b). Muscles were maintained in organ culture essentially as previously described, except that a smaller chamber was equilibrated with 95% 0, and 5% CO, daily rather than being constantly gassed. For surgery, animals were anesthetized by ketamine (40 mg/kg im), followed by pentobarbital (35 mg/kg iv). The posterior latissimus dorsi (PLD) muscle was denervated by cutting the median nerve of the brachial plexus just as it emerges from the plexus and runs beneath the scapula (Ginsborg, 1960). Denervation for 24 hr or less was accomplished simply by maintaining the dissected muscle in organ culture. RESULTS

Degradation Muscle

of AChRs

in Developing

The skeletal neuromuscular junction in fast-twitch muscles of the adult chicken is similar to the neuromuscular junction in analogous muscles of other vertebrates in most respects (the postsynaptic membrane, however, is not invaginated with deep postjunctional folds; Burden, 1977b). Autoradiography with ““I-labeled a-bungarotoxin (a-BGT) reveals that, in the PLD muscle, as in other vertebrate skeletal muscles, AChRs are highly localized to the neuromuscular junction (Fig. 1). The turnover of AChRs in the develop-

VOLUME 61, 1977

ing PLD muscle of the chick was measured by both biochemical and autoradiographic techniques. The degradation rate of AChRs was inferred from the rate of loss of bound ‘““I-labeled a-BGT. a-BGT dissociates from AChRs very slowly (t,,, = 5 days) (in rat, Berg and Hall, 1974; in chick, Burden, 1977a), and is metabolized and lost from the muscle at a faster rate only when AChRs are degraded (Berg and Hall, 1974; Devreotes and Fambrough, 1975). AChRs of PLD muscles were labeled by incubating the muscle in vitro with Ylabeled a-BGT. The muscles were maintained in organ culture, and the loss of lZ51labeled a-BGT from the tissue was assayed by measuring the appearance of radioactivity in the culture medium. The apparent degradation rate of the total muscle AChRs was determined by measuring the radioactivity remaining in the muscle at the end of the culture period and calculating the fraction remaining at each time point (Berg and Hall, 1974; Burden, 1977a). Two classes of AChRs, junctional and extrajunctional, are present in developing muscle, and the percentage of each class varies with development (Burden, 1977a). If the rates of junctional and extrajunctional receptor degradation were different, but only by several fold, intermediate rates, rather than two distinct rates, would be detected over the first few halftimes. Hence, degradation rates for total muscle receptors are referred to as apparent degradation rates. Figure 2 demonstrates that the apparent degradation rate of total muscle AChRs changes with development: The time for 50% loss shifts from 30 hr at 1 week posthatch to 43 hr at 3 weeks posthatch and, finally, to 130 hr at 5 weeks posthatch. Since the half-time, t1,2, for simple toxinreceptor dissociation in this system is approximately 120 hr (Burden, 1977a), the apparent degradation rate measured at 5 weeks posthatch is a lower limit for receptor degradation.

STEVE BURDEN

Junctional

AChR

81

Turnover

FIG. 1. Localization of AChRs. A PLD muscle from a 5-week posthatch chick was incubated with 1.0 pg/ ml of l”SI-labeled cu-BGT for 1 hr at 37°C. The muscle was washed and fixed, and single muscle fibers were dissected. (A) Bright field; (B) interference-contrast. The specific activity of the toxin was 33 Ci/mmole, and the emulsion was-exposed for 38 hr. Bar = 25 pm.

During the first few weeks after hatching, the surface area of both muscle fibers and end-plates increases, the density of junctional AChRs remains constant, and the percentage of AChRs which are extrajunctional decreases (Burden, 1977a). The shift in the rate constant for degradation could be attributed to an increase in the proportion of slowly degrading receptors or to a change of the total receptor population to intermediate degradation rates. Degradation of AChRs in Denervated Muscle Denervation of the PLD muscle from 6week posthatch chicks results in a dramatic increase in the total number of AChRs (Table 1). The extent of this increase is dependent on the duration of denervation. Likewise, the apparent rate of receptor degradation is affected by denervation: The longer the muscle has been denervated, the faster the apparent degradation rate (Fig. 3). These results are consistent with the hypothesis that, several weeks after hatching, junctional and extrajunctional receptors are degraded at different rates, and

that the intermediate rates result from an increase in the percentage of slowly degrading junctional receptors. To test this hypothesis, the degradation rate of junctional receptors was measured by autoradiographic techniques. Degradation of Autoradiography

Junctional

AChRs:

Paired PLD muscles were labeled with ““I-labeled (Y-BGT in vitro and were placed in organ culture. One muscle of each pair was maintained in organ culture for 1 day, and the other was kept in culture for 2-4 days. Single fibers were dissected from the muscle at the end of the culture period and were prepared for autoradiography. The degradation rate of junctional AChRs was determined by comparing end-plate grain densities for muscles kept in organ culture for 1 day with those maintained in culture for several days (Table 2 and Fig. 4). One week after hatching, junctional AChRs turn over rapidly (t,,, = 30 hr). Two and one-half weeks after hatching, however, the rate of junctional AChR turnover is considerably slower (t,,, 2 5 days, the rate of toxin dissociation), thus demonstrating

82

DEVELOPMENTAL

BIOLOGY

demonstrated that slowly degrading junctional AChRs do develop in the chick, and that this change occurs between 1 and 3 weeks after hatching. The number of extrajunctional receptors in embryonic muscle is regulated by synthesis, and muscle activity plays an important role in this regulation (Burden, 1977a). Activity, however, is an effective regulator of extrajunctional receptor synthesis only after a certain critical stage. Chronic paralysis of the chick embryo between Days 16 and 18 pre-

24 d

TIME

IN ORGAN

CULTURE

61, 1977

VOLUME

(ha)

FIG. 2. Degradation of receptors in organ culture. PLD muscles from 7-, 24-, and 32-day-old chicks were labeled with lz51-labeled (Y-BGT in vitro, washed, and cultured for the indicated time. At intervals, the medium was removed for counting, and fresh medium was added. At the end of the culture period, the radioactivity in the muscle was measured, and the fraction remaining bound at each time point was calculated. TABLE INCREASE

Length

1

IN THE NUMBER OF AChRs DENERVATION~

of denervation

AFTER

Toxin sites/PLD muscle (lo-l5 mole)

Not denervated 5 hr 24 hr 5 days

462 2 49 (5) 550; 610 830; 880 12.100: 9600

D PLD muscles from 6-week posthatch chicks were denervated as described in Materials and Methods. In each case, one muscle of the pair was denervated, and the other served as a control. Two muscles were denervated for each time point, and five muscles served as controls. The standard error of the mean and the number of muscles used is indicated for the controls.

a developmental change in the metabolism of junctional AChRs. DISCUSSION

AChRs at the neuromuscular junction in embryonic chick muscle turn over at the same rate as extrajunctional receptors (Burden, 1977a). The present study has

TIME IN ORGAN

CULTURE

(hrs.1

FIG. 3. Degradation of receptors from denervated muscles. Organ culture experiments were performed as described in Fig. 2. The graph illustrates the apparent degradation rates for muscles denervated for 5 hr, 24 hr, and 5 days. The muscles were from B-week posthatch chicks. TABLE DEGRADATION

Age of chick

1 week posthatch

2.5 weeks posthatch 5 weeks posthatch

2

OF JUNCTIONAL

RECEPTORS

Days in culture

Grains/ pm2 a

1

3

0.25 0.05

30

1 3

0.17 0.12

120

1

0.04 0.03

3

LI The grain densities were determined end-plates from each muscle.

Calculated t l,Z (hr)

120 for lo-12

FIG. 4. Degradation of junctional receptors. Paired PLD muscles were labeled with ‘251-labeled (Y-BCT and were maintained in organ culture as described in.Fig. 2. Fibers were then dissected and prepared for autoradiography. The end-plate grain density was determined for lo-12 fibers from each muscle. (A,B): 1 week posthatch, in culture for 1 day (A) and 3 days (B). (C,D): 2.5 weeks posthatch, in culture for 1 (C) and 3 (D) days. (E,F): 5 weeks posthatch, in culture for 1 (E) and 3 (F) days. The following half-times for degradation were calculated. One week: 30 hr; 2.5 weeks: 120 hr; and 5 weeks: 120 hr. Toxin specific activity: (A,B) 70 Ci/mmole; (C,D) 57 Wmmole; (E,F) 134 Ciimmole. The emulsion was exposed for: (A,B) 20 hr; (C,D) 18 hr; (E,F) 9 hr. Bar = 10 Frn for A-D and 20 pm for E and F.

83

84

DEVELOPMENTAL

BIOLOGY

vents the normal developmental decrease in the number of extrajunctional AChRs; paralysis between Days 12 and 13 and Days 12 and 14 does not affect the number of extrajunctional receptors. Extrajunctional receptor synthesis normally decreases at Day 16, but this is prevented by muscle paralysis. Either the type of muscle activity or the susceptibility of extrajunctional receptor synthesis to muscle activity is likely to have changed between Days 12 and Day 16 (see Boethius, 1967). Is there a similar critical stage for the change in junctional receptor turnover, and what are the signals for this change? The nerve may be necessary for the conversion of rapidly degrading receptors to slowly degrading receptors. If this were so, denervation at 1 week after hatching might prevent the appearance of slowly degrading receptors. The type and pattern of muscle activity could also be important during a critical stage so that direct stimulation of the muscle may have an effect on junctional receptor turnover. The change in turnover of junctional receptors is not correlated with a change in their density, since the density of junctional AChRs remains constant from embrvonic life through adult life (Burden, 1977a). The development of postjunctional folds is also not correlated with a change in junctional AChR turnover, since end-plates in adult PLD muscle lack this specialization (Burden, 1977b). This study and a previous study (Burden, 1977a) have emphasized that the turnover of individual receptors should be distinguished from the maintenance of receptor topography. What factors are responsible for establishing and maintaining the clustering of AChRs at the end-plate? A filamentous network beneath junctional plasma membrane, which is absent underneath extrajunctional membrane, has been observed (Ellisman et al., 1976). Junctional and extrajunctional receptors differ slightly in their isoelectric point

VOLUME

61, 1977

(Brockes and Hall, 1975). It would be of interest to examine the appearance of this filamentous network and the change in isoelectric point and to compare the timing of their development with the localization of receptors and the change in junctional receptor turnover. The present results suggest that the localization of junctional receptors and the change in their turnover can be examined separately. Receptors are concentrated at end-plates during embryonic life, but they turn over slowly only several weeks after hatching. I am grateful to Dr. G. D. Fischbach for providing laboratory facilities during the course of this work. I would like to thank Dr. Fischbach, Dr. Eric Frank, Dr. Josh Sanes, and Dr. Doju Yoshikami for their comments on the manuscript and Ms. Linda Yu for photographic assistance. The author is a National Institutes of Health Predoctoral Trainee, Grant No. GM 01874. This work was supported in part by NIH Research Grant No. NS-11160 (GDF) and American Cancer Society Institutional Grant No. IN-350 (University of Wisconsin; Dr. A.O.W. Stretton and Steve Burden). REFERENCES M. J., COHEN, M. W., and ZORYCHTA, E. (1976). Redistribution of acetylcholine receptors on cultured muscle cells. In “Sixth Annual Meeting of the Society for Neuroscience, Toronto, Canada, November 7, 1976.” BERG, D. K., and HALL, Z. W. (1974). Fate of (Ybungarotoxin bound to acetylcholine receptors of normal and denervated muscle. Science 184, 473475. BERG, D. K., and HALL, Z. W. (1975). Loss of (Ybungarotoxin from junctional and extrajunctional receptors in rat diaphragm muscle in uiuo and in organ culture. J. Physiol. (London) 252, 771-789. BEVAN, S., and STEINBACH, J. H. (1977). The distribution of a-bungarotoxin binding sites on mammalian skeletal muscle developing in uiuo. J. Physiol. (London) 267, 195-214. BBETHIUS, J. (1967). The development ofthe electromyogram in chick embryos. J. Ezp. 2001.165,419424. BROCKES, J. P., and HALL, Z. W. (1975). Acetylcholine receptors in normal and denervated rat diaphragm muscle. II. Comparison of junctional and extrajunctional receptors. Biochemistry 14, 21002106. BURDEN, S. J. (1977a). Development of the neuromuscular junction in the chick embryo: The num-

ANDERSON,

STEVE BURDEN

Junctional

ber, distribution and stability of acetylcholine receptors. Develop. Biol. 57, 317-329. BURDEN, S. J. (1977b). Thesis. University of Wisconsin. CHANG, C. C., and HUANG, M. C. (1975). Turnover of junctional and extrajunctional acetylcholine receptors of the rat diaphragm. Nature (London) 253, 643-644. DENNIS, M. J., and ORT, C. A. (1977). The distribution of acetylcholine receptors on muscle fibres of regenerating salamander limbs. J. Physiol. (Low don) 266, 765-776. DEVREOTES, P. N., and FAMBROUGH, D. M. (1975). Acetylcholine receptor turnover in membranes of developing muscle fibers. J. Cell Biol. 65,335-358. DIAMOND, J., and MILEDI, R. (1962). A study offoetal and new-born rat muscle fibers. J. Physiol. (Lendon) 162, 393-408. ELLISMAN, M. H., RASH, J. E., STAEHELIN, A., and PORTER, K. (1976). Studies of excitable membranes. II. A comparison of specializations of neuromuscular junctions and nonjunctional sarcolem-

AChR

Turnover

85

mas of mammalian fast and slow twitch muscle fibers. J. Cell Biol. 68, 752-774. FRANK, E., and FISCHBACH, G. D. (1977). ACh receptors accumulate at newly formed nerve-muscle synapses in vitro. In “Intercellular Communication: Society of General Physiologists” (Max Burger and Jay Lash, eds.), in press. GINSBORG, B ( 1960). Spontaneous activity in muscle fibers of the chick. J. Physiol. (London) 150, 707717. GORDON, T., and VRBOVA, G. (1975). Changes in chemosensitivity of developing chick muscle fibers in relation to end-plate formation. Pfliigers Arch. 360, 349-364. HOGAN, P., MARSHALL, J. M., and HALL, Z. W. (1976). Muscle activity decreases rate of degradation of a-bungarotoxin bound to extrajunctional acetylcholine receptors. Nature (London) 261, 328-330. LBMO, T., and ROSENTHAL, J. (1972). Control of ACh sensitivity by muscle activity in the rat. J. Physiol. (London) 249, 301-326.