- Email: [email protected]
Metabolic stabilization of acetylcholine receptors at newly formed neuromuscular junctions in rat
DEVELOPMENTAL
BIOLOGY
84,%i7-%d
(1981)
Metabolic Stabilization of Acetylcholine Receptors at Newly Formed Neuromuscular Junctions in Rat C. GARY REINESS AND CRISPIN B. WEINBERG Division of Neurobiology, Department of Physiology, University California 94143, and Department of Neurobiology, Harvard
San Francisco, School of Medicine, San Francisco, Medical School, 25 Shattuck St., Boston, Massachusetts, 02115
of CalZfornia,
Received June 24, 1980; accepted in revised
form October
30, 1980
The turnover of acetylcholine receptors (AChRs) was studied at developing motor endplates in embryonic rat diaphragm and at newly formed ectopic endplates in soleus muscles of adult rats. After the receptors were labeled in situ with ‘%I-a-bungarotoxin, the rate of loss of bound toxin was determined by autoradiography of single muscle fibers and was used to calculate the turnover time of AChRs. A new, convenient method for preparing large numbers of single muscle fibers is described. AChRs in extrajunctional regions of embryonic diaphragms turn over with a short half-time (24 hr) similar to that of AChRs in cultured myotubes and of extrajunctional AChRs in denervated adult muscle. AChRs in newly formed clusters in developing muscle and in ectopically innervated adult muscle also turn over with short half-times. Within a few days, however, the turnover time increases to values characteristic of adult junctional receptors (6-10 days). Transection of the nerve at newly formed ectopic endplates prevents the change. The metabolic stabilization of AChRs at motor endplates in rat muscles is thus a relatively early event in synapse formation; it coincides neither with the clustering of receptors, which precedes it, nor with the decrease in AChR channel open time which has been shown to occur postnatally.
INTRODUCTION
Acetylcholine receptors (AChRs) in normal adult vertebrate skeletal muscles are localized at the neuromuscular junction where they occur at high density (Axelsson and Thesleff, 1959; Miledi, 1960; Miledi and Potter, 1971; Hartzell and Fambrough, 1972). After denervation of adult muscles, new extrajunctional receptors appear; these occur at lower density and are diffusely distributed over the entire muscle surface (Axelsson and Thesleff, 1959; Miledi, 1960; Lee et al., 1967; Hartzell and Fambrough, 1972; Brockes and Hall, 1975a). One of the differences between these two types of AChRs is their metabolic stability. Extrajunctional receptors turn over rapidly and have a half-life in the membrane of approximately 24 hr (Berg and Hall, 1975; Chang and Huang, 1975; Heinemann et al., 1978; Reiness et al., 1978; Linden and Fambrough, 1979). Junctional receptors are much more stable and, in rat muscle, turn over with a half-time that exceeds 10 days (Reiness et al., 1978; Linden and Fambrough, 1979). In embryonic muscle fibers, AChRs are also distributed at low density over the entire muscle fiber surface (Diamond and Miledi, 1962; Bevan and Steinbach, 1977). Ingrowing neurons induce high-density clusters of AChRs at sites of neuromuscular contact (Anderson et al., 1977; Frank and Fischbach, 1979). Experiments using cell cultures of embryonic muscles (Devreotes and Fambrough, 1975) or organ cultures of muscles from newborn rats (Berg and Hall, 1975; Steinbach et al.,
1979) indicate that the nonclustered AChRs share the rapid turnover characteristic of extrajunctional receptors in denervated adult muscles. An important question is whether the clustered receptors have the rapid turnover time characteristic of extrajunctional embryonic receptors or the slower turnover time characteristic of adult junctional AChRs. In chick’muscle, clustering of receptors precedes the change in turnover time. Clustering of AChRs at new endplates occurs about 1.5 weeks before hatching (Burden, 1977a) while receptor turnover remains rapid until between 1 and 3 weeks posthatching (Burden, 1977b). Indirect measurements in newborn rat muscle, however, indicate that slowly turning over receptors are already present at birth (Berg and Hall, 1975; Steinbach et al., 1979). This is less than 1 week after clusters of AChRs form at developing rat neuromuscular junctions (Bevan and Steinbach, 1977; Braithwaite and Harris, 1979) and suggests that the processes of clustering and change in turnover time may not be distinct events in rat muscle as they are in chicken. We therefore wished to determine when, during development of neuromuscular junctions in rat muscle, metabolically stable AChRs first appear. We labeled AChRs in developing rat diaphragm with ‘?-a-bungarotoxin (lz51-a-BuTx) and used autoradiography to determine the turnover times of clustered and extrajunctional AChRs separately. Because formation of ectopic synapses in denervated adult muscle may serve as a model for endplate development, and because such
247 0012-1606/81/060247-08$02.00/o Copyright All rights
0 1981 by Academic Press, Inc. of reproduction in any form reserved.
248
DEVELOPMENTAL
BIOLOGY
VOLUME
84, 1981
Therefore, initial time points were taken at least 24 hr after injection and subsequent points were taken l-3 days later. Although, the embryos were initially paralyzed by the injection of toxin, they recovered movement within 24 hr and development did not seem to be impaired. They gained weight and developed external characteristics at the same rate as normal embryos and, after birth, were indistinguishable from normal pups. Labeling of ectopic endplates. Ectopic endplates were produced on soleus muscles as described by Frank et al. MATERIALS AND METHODS (1975b). The fibular branch of the sciatic nerve was exMaterials. Sprague-Dawley rats were used in all ex- posed in the ankle and implanted on the proximal endperiments. They were obtained either from Simonsen plate-free region of the soleus muscle of a 150 to 200Laboratories, Gilroy, California, from Charles River g rat. After 2 to 3 weeks the soleus muscle was denerLaboratories, Wilmington, Massachusetts, or from a vated by resection of the tibia1 branch of the sciatic breeding colony of Charles River rats maintained by nerve in the thigh. This enabled the implanted fibular Dr. M. Dennis at the University of California, San Franbranch to form functional contacts with the soleus cisco. Pregnancies were timed from the appearance of muscle 2 l/2 to 3 days after denervation (Lomo and a copulation plug and ages confirmed by matching emSlater, 1976, 1978). bryo weights to published tables (Angulo y Gonzalez, At times ranging from 2 to 40 days after denervation, 1932). Pups were born on the 22nd day of gestation. 1251- the AChRs of leg muscles were labeled by injection of cu-BuTx with a specific activity of 100 cpm/fmole (de- lWI-a-BuTx into the femoral artery of rats anesthetized termined on a Beckman Biogamma II counter at 70% with ether. A dose of lo-12 pg/lOO g body wt was sufefficiency) was prepared as described previously (Berg ficient to bind BO-90% of the AChRs in the leg without et al., 1972) and stored in 20 mM sodium phosphate, pH morbidity. In control experiments on normal muscles, 7.4, with 1 mg/ml bovine serum albumin. the mean number of grains per endplate varied by Labeling of embos. To measure the turnover of f 10% from animal to animal. Free ‘251-~-B~T~ in the bloodstream fell to less than 20% of its initial level 3 AChRs during development we labeled rat embryos hr after injection and this was defined as the time of with ‘%I-a-BuTx. Pregnant females were anesthetized with ether, the horns of the uterus were externalized, labeling. Rats were killed various times after labeling and each embryo was injected im with 0.5-1.0 pg of 1251- and the soleus muscle was removed. a-BuTx in a volume of 3-5 ~1 using a lo-p1 Hamilton Preparation of single jibers. Labeled muscles were syringe. We are indebted to Dr. A. J. Harris for dempinned to Sylgard-lined dishes, washed with saline, and onstrating this technique of intrauterine injection fixed in 0.8% glutaraldehyde and 2% formaldehyde in to us. 150 mM NaCl, 3 mM CaC12, 30 mM Hepes buffer at 4°C At appropriate times after injection the mothers overnight. A patch of muscle 5 mm square was dissected were anesthetized and three or four embryos were re- from the central portion of the diaphragm. For ectopic moved. The embryos were weighed and the diaphragms endplates, a segment of the soleus under the foreign dissected and processed for autoradiography as de- nerve and devoid of the original endplates was used. To scribed below. Postanatal data were obtained by re- obtain single fibers, fixed muscle segments were homoving diaphragms from pups. mogenized in lo-15 ml of distilled water in a VirTis In these experiments it was important that free 1251- homogenizer at setting 30-50 for several seconds and a-BuTx be rapidly removed so that relabeling of the then at setting 20 for lo-30 sec. This procedure usually receptors would not occur during the period of meabroke the muscle into long (200- to 2000-pm) single fisurement. To determine the rate of toxin removal we bers (Fig. 1). This method is more rapid and efficient injected litters at embryonic day (ED) 16 or ED 20. Six than the customary method of teasing out indivdual hours after the injection, the radioactivity in bulk car- muscle fibers (Bevan and Steinbach, 1977; Burden, cass muscle had fallen to 10% of the original level and 1977a). by 24 hr it had declined to less than 4% of the original Autoradiographv. Gelatin-coated slides containing a value. Over 70% of the remaining radioactivity was in few drops of a fiber suspension were air dried, dipped the form of toxin-receptor complex since, after soluin a mixture of two parts Kodak NTB-2 emulsion to one bilization in detergent, it was retained by DEAE filters part 3% glycerol at 4O”C, and exposed at 4°C for 8 hr and was precipitated by an antiserum to rat AChRs. to 10 days. The slides were developed for 2 min at room synapses are more accessible to experimental manipulation, we have also examined the turnover properties of AChRs during the formation of ectopic synapses in rat soleus muscle. Our results show that in both cases AChRs in newly formed clusters turn over rapidly; within 5 days, however, their turnover time changes to that of adult junctional receptors. Furthermore, we show that the continued presence of the nerve is necessary to effect this change in stability of clustered AChRs at ectopic endplates.
labilization
FIG. 1. Photomicrograph of a slide containing muscle fibers from a rat diaphragm (ED 21) prepared for autoradiography. Bar = 1 mm.
temperature (20-22°C) in a Kodak D-19 developer made up in 1% glycerol, fixed, dried, and mounted with Permount (Fisher Chemical Co.). Silver grains were counted under darkfield illumination using a Zeiss microscope with 10X eyepieces and a 40X Neofluar objective. A drawing tube was used to trace an area of muscle fiber onto graph paper which had been calibrated using a stage micrometer. It was assumed that only radioactivity on the surface of the muscle in contact with emulsion generated grains. Grain densities over unlabeled fibers or over extrajunctional regions of labeled normal adult muscles were not significantly above background grain densities. Background grain densities, measured near each muscle fiber, were not more than 10% of the grain density within clusters. No corrections were made for curvature of the fibers since only clusters that were viewed en face were counted. For extraCalculation of half-times for turner. junctional regions, the grain density was calculated by counting the number of silver grains in at least 1000 l.cm2 of each fiber surface and subtracting the background grain density. Eight fibers were analyzed per muscle and one to four muscles were examined for each time point. The mean density was plotted on semilogarithmic coordinates as a function of the time after labeling, and the half-life was determined from the slope. For clustered receptors, the number of grains per cluster was counted on four to eight fibers in each muscle and corrected for background grain density. Clusters were readily identified as areas whose grain density after subtraction of background exceeded that of surrounding areas by approximately an order of magnitude. Identification and quantitation of grains in clusters by two independent observers yielded values differing by less than 10%. Further, determination of cluster size at ectopic synapses using rhodamine-labeled a-bungarotoxin gave sizes similar to those obtained by autoradiography (Weinberg et al., 1981b). The total number of grains per cluster rather than density
of ACh
249
Receptors
was used to determine changes in the number of labeled AChRs in the cluster because significant changes in the area occupied by labeled AChRs occur following a pulse label (Weinberg, et aZ., 1981a). For ectopic synapses the mean number of grains per cluster was plotted as a function of the time after labeling and the half-time for turnover obtained as above. For synapses in developing muscle, one litter was used for each experiment. Because it was not feasible to remove embryos on a daily basis without endangering the mother, groups of two to four embryos were taken from each litter at two time points, 48-72 hr apart. In each embryo the number of grains per cluster was determined for five to eight fibers from the diaphragm. The SEM of the grains per cluster in each animal was about 10% of the mean value. The mean half-life for turnover within the litter was determined by taking the mean of the half-times calculated for each muscle taken at an early time paired with each muscle taken at a late time, assuming that the loss of radioactivity was exponential. The SEM of these values (shown in Fig. 4) represents the variability among diaphragms in a litter. An alternative method, in which all fibers from animals taken at the same time were pooled, and the mean and SEM of these values used to compute the mean and SEM of half-lives for turnover gave quantitatively similar results (data not shown). Additional methods. In some control experiments slides were stained for acetylcholinesterase through the developed emulsion for 15-30 min at room temperature (Karnovsky and Roots, 1964). Binding of ‘%I-a-BuTxAChR complexes to DEAE filters and immunoprecipitation were performed as described previously (Brockes and Hall, 197533; Weinberg and Hall, 1979). RESULTS
Developing
Muscles
We determined the apparent turnover time of AChRs in developing rat diaphragms by labeling embryos with 1251-~-B~T~ and using autoradiography to measure the loss of radioactivity from receptor clusters and from extrajunctional regions of muscle fibers. In extrajunctional regions radioactive toxin was lost rapidly from all muscles. The results of an experiment in which embryos were injected on ED 21 and the density of grains in extrajunctional regions of muscle fibers was measured 1, 2, and 3 days later are shown in Fig. 2. The grain density decreased exponentially with a half-time of 24 hr. In other experiments we determined grain density at only two times and calculated a halftime assuming that the loss of toxin was exponential. The half-times of toxin loss from extrajunctional re-
250
0.4L
1 0
I 48 Time after labeling
I 96 (hr)
FIG. 2. Extrajunctional grain density on developing muscle fibers from rat diaphragm as a function of time after labeling with ‘251-(uBuTx. The litter was injected in utero on ED 21 and the first point taken 24 hr later (at birth). Density was measured on 16-24 fibers from two or three diaphragms at each time point. Points represent mean grain density + SEM.
gions of embryonic muscle did not change between ED 18-22. This half-time of 24 + 2 hr (mean + SEM; n = 5) is in the same range as the values we obtained from denervated adult diaphragm (17 f 2 hr) and soleus (25 f 8 hr) muscles. These values also fall within the range of estimates of the turnover time for extrajunctional receptors made by other methods in denervated adult muscles (Berg and Hall, 1975; Chang and Huang, 1975) and in myotubes in cell culture (Devreotes and Fambrough, 1975). In diaphragms, clusters of AChRs first appeared at ED 15-16 (Bevan and Steinbach, 1977; Braithwaite and Harris, 1979). Although the grain clusters observed in young fibers were more diffuse than the sharply defined, dense clusters observed later, clustered receptors were always easily distinguished from those in extrajunctional regions (see Fig. 3). In general, only one cluster was seen per fiber; however, in fibers from young
muscles (ED 18 or earlier) a group of two to four small clusters was occasionally seen (see also Bennett and Pettigrew, 1974; Bevan and Steinbach, 1977) and these were excluded from turnover measurements. Over 80% of the receptor clusters at ED 16, and over 90% in older embryos, coincided with sites of acetylcholinesterase staining. Since acetylcholinesterase characterizes sites of neuromuscular transmission (Bennett and Pettigrew, 1974) we conclude that the clustered AChRs are at neuromuscular junctions. As illustrated by the autoradiographs shown in Fig. 3, ‘251-a-BuTx was lost rapidly from clusters of AChRs at the earliest times examined, but within 4 days the toxin was lost much more slowly. To characterize the rate of loss during development, we measured grain densities in clusters at two times after labeling and from these calculated apparent half-times of turnover of AChRs in clusters. For convenience, we assumed that toxin loss occurred with a single exponential process, although this may not be the case at intermediate times. The results of autoradiographic measurements of such apparent turnover times of clustered AChRs during development are summarized in Fig. 4. On ED 17 and 18, toxin was lost from clusters with a half-time of 3235 hr, which is similar to that obtained for extrajunctional receptors. By ED 21, the half-time for toxin loss exceeded 145 hr in four out of five litters; these values were not distinguishable from those obtained for junctional AChRs in adult diaphragm. Although these measurements cannot distinguish between a change in turnover rate for all clustered receptors and a change in the proportion of slowly and rapidly turning over receptors within clusters, it is clear that the AChRs at endplates in embryonic rat muscle undergo a marked increase in turnover time between ED 18 and 21, whereas extrajunctional receptor turnover times remain low throughout development.
FIG. 3. Dark-field photomicrographs of autoradiographs of muscle fibers from developing rat diaphragms. Grain clusters on fibers from litter-mates injected with ‘z51-a-BuTx on ED 17 and prepared (a) 1 day or (b) 3 days later, or from littermates injected on ED 20 and prepared (c) 2 days later (at birth) or (d) 4 days later. Bar = 20 pm. The single fibers shown in each micrograph are oriented horizontally and are approximately the same width as the micrograph.
REINESS
EJR
ED 17
ED IS
ED 19
ED 20
21
21
ED 21
AND WEINBERG
Birth
PD 8
Adult JR
AGE
FIG. 4. Half-lives for turnover of AChRs clustered at developing endplates in rat diaphragm. Each bar represents results from a separate litter. Lower axis gives age of animals at initial time point. Shown for comparison are half-lives measured for embryonic extrajunctional receptors (EJR) and junctional receptors of S&day postnatal (PD 8) or of adult rats. At each time point, a total of lo-20 clusters from diaphragms of two to four animals were analyzed except for the ED 1’7, ED 19, and one of the ED 20 litters where only a single animal (5-8 clusters) was available at the second time point. Bar represents SEM. The method used to determine turnover times in these experiments could not distinguish values in excess of 150 hr (see Materials and Methods).
Ectopic Endplates Cutting the original nerve to a muscle enables a foreign nerve previously implanted in an endplate-free zone to make new endplates (Elsberg, 1917). We used these ectopic endplates in adult muscle as a second system in which to study the change in AChR turnover time that occurs during synaptogenesis. AChRs were labeled by injection of a sublethal dose of 1251-a-BuTx that bound about 90% of the receptors in the rat soleus muscle. Muscles were removed at various times after injection and loss of radioactivity was determined by counting grains per cluster in autoradiographs of single fibers. In control experiments on normal soleus muscles the half-time for toxin loss at adult endplates was 10 f 1.5 days, which is similar to values obtained in soleus muscles (Linden and Fambrough, 1979) or rat diaphragms (Reiness et aZ., 1978) maintained in organ culture, or by autoradiography of adult diaphragm (Fig. 4). In agreement with earlier results (Berg and Hall, 1975; Chang and Huang, 1975; Linden and Fambrough, 1979) the extrajunctional AChRs that appeared after denervation turned over much more rapidly with a half-time around 1 day (25 f 8 hr).
Stabilization
251
of ACh Receptors
In muscles receiving ectopic innervation, clusters were first seen 2 days after denervation and their size and grain density increased gradually over the next few weeks. Such clusters were substantially larger than those seen by Ko et al. (1977) in denervated rat soleus muscles (Weinberg et aZ., 1981b), and were not seen in the distal nerve-free segment of ectopically innervated muscles nor in denervated soleus muscles lacking an implanted foreign nerve. The turnover time of receptors in these clusters was determined by plotting the number of grains per ectopic endplate vs time after labeling. The results of these experiments are shown in Fig. 5. Two days after denervation, toxin was lost exponentially with a half-time of 24 f 5 hr. At later times toxin was lost exponentially with longer half-times (7.5 -+ 1.5 to 10 f 1.5 days for muscles labeled 6,1’7, and 40 days after denervation). Thus the rate of toxin loss from clustered AChRs at ectopic endplates changed between 2 and 6 days after denervation from a rapid value characteristic of extrajunctional receptors to a slow value characteristic of junctional receptors. In order to determine if the change in rate of toxin loss requires the presence of the nerve, we cut the for-
’ O”
BLIP
17 days
6 days
4 days
2 days
Time after
labeling
(hr)
FIG. 5. Loss of ‘=I-cr-BuTx from newly formed ectopic endplates on rat soleus muscles. Ectopic innervation was produced by cutting the original soleus nerve 2-3 weeks after implanting a foreign nerve on an endplate-free region of the muscle. AChRs were labeled in tivo at ages ranging from 2 to 40 days after cutting the original nerve. Toxin bound to eetopic endplates was determined by counting the grains per cluster on autoradiographs of single muscle fibers. The values obtained were corrected for the extrajunctional grain density and normalized to an exposure time of 1 week with a specific activity of 100 cpm/fmole. The number of grains per cluster is plotted as a function of the time after the muscle was labeled. Each point represents the mean & SEM of three to eight (usually four to six) endplates.
DEVELOPMENTALBIOLOGY
252
eign nerve 3 days after denervating the soleus. At this time clusters have formed underneath the ectopic nerve but still consist primarily of rapidly turning over receptors. Three days later we labeled the muscles with ‘%I-cu-BuTx. Clusters were still seen at the site of ectopic innervation in these muscles which shows that maintenance of newly formed clusters does not require the presence of the foreign nerve. The number of AChRs in these clusters was unchanged from the number when the foreign nerve was cut, but the clusters were about 30% larger and more diffuse than those seen before the foreign nerve had been cut. The loss of toxin from these denervated ectopic endplates is shown in Fig. 6. Toxin was lost exponentially with a half-time of 42 + 14 hr. Thus toxin was lost more rapidly from denervated ectopic endplates than from those in which the foreign nerve remained intact (t112 = 7.5 + 1.5 days).
DISCUSSION
We have found that ‘251-a-BuTx is rapidly lost from newly formed synapses both during normal development and during ectopic innervation of adult muscle (tl,z g 1 day). Within 5 days of cluster formation, however, the toxin is lost, as from adult endplates, at a much slower rate (tl,z = 6-10 days). Previous work has shown that ‘=I-a-BuTx binds specifically to AChRs in skeletal muscle (Chang and Lee, 1963; Miledi and Potter, 1971; Berg et al., 1972) and that at least some of the loss of radioactivity from the muscle reflects internalization and degradation of the toxin-receptor complex (Berg and Hall, 1975; Devreotes and Fambrough, 1975; Libby et al., 1980). Radioactivity is lost from muscle both by turnover of the AChR-‘%I-cw-BuTx complex and by dissociation 50
5Ll 0
I 90
48 Time after labeling
(hr)
FIG. 6. Loss of r&I-a-BuTx from denervated ectopic endplates. Ectopic endplates were denervated by resecting the foreign nerve 3 days after the original nerve had been cut. Three days later (6 days after cutting the original nerve) AChRs were labeled with ‘%I-a-BuTx. Toxin bound to the denervated ectopic endplates was determined as in Fig. 5. Each point represents the mean + SEM of four to six endplates.
VOLUME
84, 1981
of the toxin from the receptor. Since the half-time for dissociation of lzT-~-BuTx from AChR of adult muscles in organ culture is about 2 weeks (Reiness et al., 1978; Linden and Fambrough, 1979), this process does not contribute appreciably to loss of toxin from rapidly turning over receptors (t1,2 zz5 days), but may constitute a substantial fraction of toxin loss from receptors which turn over more slowly. Thus our measurements of toxin loss by autoradiography are likely to underestimate the turnover time for more stable receptors. Diffusion of labeled AChRs out of clusters could increase the apparent rate of turnover, and might account for the initially rapid rate of toxin loss. Our results do not allow us to distinguish the contributions of diffusion and degradation to the rate of toxin loss.. While no data are available about the diffusion coefficient of AChRs in developing endplates, AChRs which cluster on uninnervated cultured rat myotubes are not freely diffusible (Axelrod et al., 1976). If the same is true in viva, diffusion of AChRs away from the clusters would not account for the initially rapid rate of loss. Accumulation of toxin by diffusion of labeled receptors into a cluster would decrease the apparent rate of loss of toxin. Thus the change in turnover that we describe might be due to accumulation of receptors by diffusion. We followed the calculation of Edwards and Frisch (1976) for the accumulation of receptors at an endplate. Using our measurements of cluster size, extrajunctional grain density, and turnover time, and Axelrod et uZ.‘s, (1976) measurement of 6 X 10-u cm21 set for the diffusion constant of nonclustered receptors on cultured rat myotubes, we found that less than 10% of the total label in a cluster would be added by diffusion during 24 hr. For a cluster of rapidly turning over receptors this would increase the apparent turnover time by less than 5 hr. Thus accumulation of AChRs by diffusion could not account for the increase in turnover time that we observed at new endplates. We found that AChRs in clusters at new synapses in rat muscles initially have a short half-life in the membrane that is comparable to that of extrajunctional AChRs. The rapid loss of AChRs could reflect the disappearance of the clusters themselves; however, clusters at newly formed nerve-muscle synapses in vitro persist (Frank and Fischbach, 1979). Thus the rapid loss probably reflects turnover of AChRs within the clusters. Clustering of AChRs and their metabolic stabilization are therefore independent processes in rat muscle indicating that the two processes have different mechanisms. Similar results were obtained in developing chick muscle by Burden (1977a). Clustered receptors with short half-times have also been found at long-term denervated adult endplates (Brett and Younkin, 1979; Loring and Salpeter, 1980).
REINESS AND WEINBERG
Burden (1977b) found in chicken posterior latissimus dorsi muscle that more than 2 l/2 weeks elapsed between the formation of clusters and the change in receptor turnover time. In rats, in contrast, the change to a long turnover time occurs a few days after clustering at an endplate. In rat embryos the turnover time increased to adult values between ED 17 and ED 21. This is consistent with earlier results which suggested that slowly turning over AChRs are present in neonatal (ED 22) rat diaphragms (Berg and Hall, 19’75; Steinbach et al., 1979). A similar time course was seen at ectopic endplates where most of the change in turnover time occurred between the third and the sixth days after cutting the original nerve. We showed that the continued presence of the nerve is required for the metabolic stabilization of AChRs and, as at mature synapses (Frank et al., 1975a), the nerve is not required for the maintenance of receptor clusters. The mechanism by which this stabilization takes place is not clear. The rapidly turning over receptors may either be replaced by or converted to the more slowly turning over form. Bloch and Steinbach (1979) have reported that clusters of AChRs at developing neuromuscular contacts can initially be readily dispersed by removal of Ca2+ but that receptors at older endplates are resistant to such treatment. This resistance to dispersal within the membrane may correspond to the metabolic stabilization described in this paper. Several studies indicate that the mean channel open time of AChRs at motor endplates changes later during development than the change in metabolic stability that we have described (Sakmann and Brenner, 1978; Fischbath and Schuetze, 1980). The relationship of these changes in physiological properties during development to changes in the molecular form of the AChR is not clear. The time course of the development of molecular properties such as differences in isoelectric point (Brockes and Hall, 1975c) and in antigenic determinants (Almon and Appel, 1975; Weinberg and Hall, 1979) remains to be established; however, since the changes in physiological properties follow different time courses, a single molecular alteration could not account for all of them. The results at ectopic endplates parallel those at developing embryonic endplates. In both cases clusters of rapidly turning over receptors are formed, and within 5 days the receptors are metabolically stable. Ectopic endplates offer particular advantages for the study of neuromuscular synapse formation since they mature relatively synchronously and are readily accessible to experimental manipulation. In summary, there are at least three distinct phases in the appearance of mature, junctional properties of
Stabilization
of ACh Receptors
253
AChRs during the formation of new endplates. First, AChRs become clustered at sites of nerve-muscle contact. During the subsequent 4-5 days their turnover time increases at least 5- to lo-fold. Finally, l-2 weeks later, the mean channel open time changes during development to that characteristic of adult AChRs (Sakmann and Brenner, 1978; Fischbach and Schuetze, 1980). We thank Dr. Zach W. Hall, in whose laboratory at the University of California, San Francisco, these experiments were conducted, for his advice and encouragement, and for helping to develop the technique for single fiber preparation. We also thank Miu Lam for skillful technical assistance and Liz Neville and Veronica Oliva for assistance in preparation of the manuscript. This work was supported by postdoctoral fellowships from the Muscular Dystrophy Association (MDA) and the National Institutes of Health (NIH) to C.G.R. a predoctoral fellowship from the National Science Foundation to C.B.W., and grants from MDA and NIH to Z. W. Hall.
REFERENCES ALMON, R. R., and APPEL, S. H. (1975). Interaction of myasthenic serum globulin with the acetylcholine receptor. Biochim. Biophys. Acta 393,66-W. ANDERSON, M. J., COHEN, M. W., and ZORYCHTA, E. (1977). Effects of innervation on the distribution of acetylcholine receptors on cultured muscle cells. J. Physiol. 268, 731-756. ANGULO Y GONZALEZ, A. W. (1932). The prenatal growth of the albino rat. Anat. Rec. 52,117-138. AXELROD, D., RAVDIN, P., KAPPEL, D. E., SCHLESSINGER, J., WEBB, W. W., ELSON, E. L., and PODLESKI, T. (1976). Lateral motion of fluorescently labeled acetylcholine receptors in membranes of developing muscle fibers. Proc. Nat. Acad Sci. USA 73,4594-4598. AXELSSON, J., and THESLEFF, S. (1959). A study of supersensitivity in mammalian muscle. J. Physiol. 147, 178-193. BENNETT, M. R., and PETTIGREW, A. G. (1974). The formation of synapses in striated muscle during development. J. Physiol. 241.515545. BERG, D. K., KELLY, R. B., SARGENT, P. B., WILLIAMSON, P., and HALL, Z. W. (1972). Binding of a-bungarotoxin to acetylcholine receptors in mammalian muscle. Proc. Nat. Acad. Sci. U&l 69,147-151. BERG, D. K., and HALL, Z. W. (1975). Loss of a-bungarotoxin from junctional and extrajunctional acetylcholine receptors in rat diaphragm muscle in viva and in organ culture. J. Physiol. 252, 771789. 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. (1979). The effect of Ca++-deprivation on accumulations of acetylcholine receptor at the developing neuromuscular junction. Sot. Neurosci. Abstr. 5,476. BRAITHWAITE, A. W., and HARRIS, A. J. (1979). Neural influence on acetylcholine receptor clusters during embryonic development of skeletal muscles. Nature (London) 279, 549-551. BRETT, R. S., and YOUNKIN, S. G. (1979). Accelerated degradation of junctional a-bungarotoxin-acetylcholine receptor complexes in denervated rat diaphragm. Sot. Neurosci. Abstr. 5, 765. BROCKES, J. P., and HALL, Z. W. (1975a). Synthesis of acetylcholine receptor by denervated rat diaphragm muscle. Proc. Nat. Acad. Sci. USA 72,1368-1372. BROCKES, J. P., and HALL, Z. W. (1975b). Acetylcholine receptors in
254
DEVELOPMENTAL BIOLOC:Y
normal and denervated rat muscle. I. Purification and interaction with [‘Z51]-a-bungarotoxin. Biochemistry 14, 2092-2099. BROCKES, J. P., and HALL, Z. W. (1975c). Acetylcholine receptors in normal and denervated rat muscle. II. Comparison of junctional and extrajunctional receptors. Biochemistry 14,2100-2106. BURDEN, S. (1977a). Development of the neuromuscular junction in the chick embryo: the number, distribution, and stability of acetylcholine receptors. Develop. Biol. 57.317-329. BURDEN, S. (1977b). Acetyleholine receptors at the neuromuscular junction: developmental change in receptor turnover. Develop. Biol. 61, 79-85. CHANG, C. C., and HUANG, M. C. (1975). Turnover of junctional and extrajunctional acetylcholine receptors of the rat diaphragm. Nature (London) 253,643-644. CHANG, C. C., and LEE, C. Y. (1963). Isolation of neurotoxins from the venom of Bungarus multicinctus and their modes of neuromuscular blocking action. Arch. Int. Pharmacodyn. 144,241~257. 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 of foetal and new-born rat muscle fibres. J. Physiol. 162, 393-408. EDWARDS, C., and FRISCH, H. L. (1976). A model for the localization of acetylcholine receptors at the muscle endplate. J. Neurobiol. 7, 377-381. 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. FISCHBACH, G. D., and SCHUETZE, S. M. (1980). A postnatal decrease in acetylcholine channel open time at rat endplates. J. Physiol. 303, 125-137. FRANK, E., and FISCHBACH, G. D. (1979). Early events in neuromuscular junction formation in vitro. J. Cell. BioZ. 83.143-158. FRANK, E., GAUTVIK, K., and SOMMERSCHILD, H. (1975a). Cholinergic receptors at denervated mammalian motor end-plates. Acta Physiol. Stand. 95, 66-76. FRANK, E., JANSEN, J. K. S., LIMO, T., and WESTGAARD, R. H. (1975b). The interaction between foreign and original motor nerves innervating the soleus muscle of rats. J. Physiol. 247, 725-743. HARTZELL, H. C., and FAMBROUGH, D. M. (1972). Acetylcholine receptors: Distribution and extrajunctional density in rat diaphragm after denervation correlated with aeetylcholine sensitivity. J. Gen. Physiol. 60, 248-262. HEINEMANN, S., MERLIE, J., and LINDSTROM, J. (1978). Modulation of acetylcholine receptor in rat diaphragm by anti-receptor sera. Nature (London) 274, 65-68. KARNOVSKY, M. F., and ROOTS, L. (1964). A “direct-coloring” thio-
VOLUME 84, 1981 choline method for cholinesterase. J. Histochem. Cytochem. 12,219-
221. 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. LEE, C. Y., TSENG, L. F., and CHIU, T. H. (1967). Influence of denervation on localization of neurotoxins from elapid venoms in rat diaphragm. Nature fLondcm) 215, 1177-1178. LIBBY, P., BURSZTAJN, S., and GOLDBERG, A. L. (1980). Degradation of the acetylcholine receptor in cultured muscle cells: Selective inhibitors and the fate of undegraded receptors. Cell 19.481-491. LINDEN, D. C., and FAMBROUGH, D. M. (1979). Biosynthesis and degradation of acetylcholine receptors in rat skeletal muscles. Effects of electrical stimulation. Neuroscience 4, 527-538. 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. LORING, R. H., and SALPETER, M. M. (1980). Denervation increases turnover rate of junctional acetylcholine receptors. Proc. Nat. Acad. Sci. USA 77, 2293-2297. MILEDI, R. (1960). The acetylcholine sensitivity of frog muscle fibres after complete or partial denervation. J. Physiol. 151, l-23. MILEDI, R., and POTTER, L. T. (1971). Acetylcholine receptors in muscle fibres. Nature (London) 233,599~603. REINESS, C. G., WEINBERG, C. B., and HALL, Z. W. (1978). Antibody to acetylcholine receptor increases degradation of junctional and extrajunctional receptors in adult muscle. Nature (London) 274,6870. SAKMANN, B., and BRENNER, H. R. (1978). Change in synaptic channel gating during neuromuscular development. Nature (London) 276, 401-402.
STEINBACH, J. H., MERLIE, J., HEINEMANN, S., and BLOCH, R. (1979). Degradation of junctional acetylcholine receptors by developing rat skeletal muscle. Proc. Nat. Acad. Sci. USA 76, 3547-3551. WEINBERG, C. B., and HALL, Z. W. (1979). Antibodies from patients with myasthenia gravis recognize determinants unique to extrajunctional acetylcholine receptors. Proc. Nat. Acad. Sci. USA 76, 504-508. WEINBERG, C. B., REINESS, C. G., AND HALL, E. W. (1981a). TOPOgraphical segregation of old and new acetylcholine receptors at developing eetopic endplates in adult rat muscle. J. Cell Biol. 88, 215-218. WEINBERG, C. B., SANES, J. R., AND HALL, R. W. (1981b). Formation of neuromuscular junctions in adult rats: accumulation of acetylcholine receptors, acetylcholinesterase, and components of synaptic basal lamina. Develop. Biol. 84,255-266.
BIOLOGY
84,%i7-%d
(1981)
Metabolic Stabilization of Acetylcholine Receptors at Newly Formed Neuromuscular Junctions in Rat C. GARY REINESS AND CRISPIN B. WEINBERG Division of Neurobiology, Department of Physiology, University California 94143, and Department of Neurobiology, Harvard
San Francisco, School of Medicine, San Francisco, Medical School, 25 Shattuck St., Boston, Massachusetts, 02115
of CalZfornia,
Received June 24, 1980; accepted in revised
form October
30, 1980
The turnover of acetylcholine receptors (AChRs) was studied at developing motor endplates in embryonic rat diaphragm and at newly formed ectopic endplates in soleus muscles of adult rats. After the receptors were labeled in situ with ‘%I-a-bungarotoxin, the rate of loss of bound toxin was determined by autoradiography of single muscle fibers and was used to calculate the turnover time of AChRs. A new, convenient method for preparing large numbers of single muscle fibers is described. AChRs in extrajunctional regions of embryonic diaphragms turn over with a short half-time (24 hr) similar to that of AChRs in cultured myotubes and of extrajunctional AChRs in denervated adult muscle. AChRs in newly formed clusters in developing muscle and in ectopically innervated adult muscle also turn over with short half-times. Within a few days, however, the turnover time increases to values characteristic of adult junctional receptors (6-10 days). Transection of the nerve at newly formed ectopic endplates prevents the change. The metabolic stabilization of AChRs at motor endplates in rat muscles is thus a relatively early event in synapse formation; it coincides neither with the clustering of receptors, which precedes it, nor with the decrease in AChR channel open time which has been shown to occur postnatally.
INTRODUCTION
Acetylcholine receptors (AChRs) in normal adult vertebrate skeletal muscles are localized at the neuromuscular junction where they occur at high density (Axelsson and Thesleff, 1959; Miledi, 1960; Miledi and Potter, 1971; Hartzell and Fambrough, 1972). After denervation of adult muscles, new extrajunctional receptors appear; these occur at lower density and are diffusely distributed over the entire muscle surface (Axelsson and Thesleff, 1959; Miledi, 1960; Lee et al., 1967; Hartzell and Fambrough, 1972; Brockes and Hall, 1975a). One of the differences between these two types of AChRs is their metabolic stability. Extrajunctional receptors turn over rapidly and have a half-life in the membrane of approximately 24 hr (Berg and Hall, 1975; Chang and Huang, 1975; Heinemann et al., 1978; Reiness et al., 1978; Linden and Fambrough, 1979). Junctional receptors are much more stable and, in rat muscle, turn over with a half-time that exceeds 10 days (Reiness et al., 1978; Linden and Fambrough, 1979). In embryonic muscle fibers, AChRs are also distributed at low density over the entire muscle fiber surface (Diamond and Miledi, 1962; Bevan and Steinbach, 1977). Ingrowing neurons induce high-density clusters of AChRs at sites of neuromuscular contact (Anderson et al., 1977; Frank and Fischbach, 1979). Experiments using cell cultures of embryonic muscles (Devreotes and Fambrough, 1975) or organ cultures of muscles from newborn rats (Berg and Hall, 1975; Steinbach et al.,
1979) indicate that the nonclustered AChRs share the rapid turnover characteristic of extrajunctional receptors in denervated adult muscles. An important question is whether the clustered receptors have the rapid turnover time characteristic of extrajunctional embryonic receptors or the slower turnover time characteristic of adult junctional AChRs. In chick’muscle, clustering of receptors precedes the change in turnover time. Clustering of AChRs at new endplates occurs about 1.5 weeks before hatching (Burden, 1977a) while receptor turnover remains rapid until between 1 and 3 weeks posthatching (Burden, 1977b). Indirect measurements in newborn rat muscle, however, indicate that slowly turning over receptors are already present at birth (Berg and Hall, 1975; Steinbach et al., 1979). This is less than 1 week after clusters of AChRs form at developing rat neuromuscular junctions (Bevan and Steinbach, 1977; Braithwaite and Harris, 1979) and suggests that the processes of clustering and change in turnover time may not be distinct events in rat muscle as they are in chicken. We therefore wished to determine when, during development of neuromuscular junctions in rat muscle, metabolically stable AChRs first appear. We labeled AChRs in developing rat diaphragm with ‘?-a-bungarotoxin (lz51-a-BuTx) and used autoradiography to determine the turnover times of clustered and extrajunctional AChRs separately. Because formation of ectopic synapses in denervated adult muscle may serve as a model for endplate development, and because such
247 0012-1606/81/060247-08$02.00/o Copyright All rights
0 1981 by Academic Press, Inc. of reproduction in any form reserved.
248
DEVELOPMENTAL
BIOLOGY
VOLUME
84, 1981
Therefore, initial time points were taken at least 24 hr after injection and subsequent points were taken l-3 days later. Although, the embryos were initially paralyzed by the injection of toxin, they recovered movement within 24 hr and development did not seem to be impaired. They gained weight and developed external characteristics at the same rate as normal embryos and, after birth, were indistinguishable from normal pups. Labeling of ectopic endplates. Ectopic endplates were produced on soleus muscles as described by Frank et al. MATERIALS AND METHODS (1975b). The fibular branch of the sciatic nerve was exMaterials. Sprague-Dawley rats were used in all ex- posed in the ankle and implanted on the proximal endperiments. They were obtained either from Simonsen plate-free region of the soleus muscle of a 150 to 200Laboratories, Gilroy, California, from Charles River g rat. After 2 to 3 weeks the soleus muscle was denerLaboratories, Wilmington, Massachusetts, or from a vated by resection of the tibia1 branch of the sciatic breeding colony of Charles River rats maintained by nerve in the thigh. This enabled the implanted fibular Dr. M. Dennis at the University of California, San Franbranch to form functional contacts with the soleus cisco. Pregnancies were timed from the appearance of muscle 2 l/2 to 3 days after denervation (Lomo and a copulation plug and ages confirmed by matching emSlater, 1976, 1978). bryo weights to published tables (Angulo y Gonzalez, At times ranging from 2 to 40 days after denervation, 1932). Pups were born on the 22nd day of gestation. 1251- the AChRs of leg muscles were labeled by injection of cu-BuTx with a specific activity of 100 cpm/fmole (de- lWI-a-BuTx into the femoral artery of rats anesthetized termined on a Beckman Biogamma II counter at 70% with ether. A dose of lo-12 pg/lOO g body wt was sufefficiency) was prepared as described previously (Berg ficient to bind BO-90% of the AChRs in the leg without et al., 1972) and stored in 20 mM sodium phosphate, pH morbidity. In control experiments on normal muscles, 7.4, with 1 mg/ml bovine serum albumin. the mean number of grains per endplate varied by Labeling of embos. To measure the turnover of f 10% from animal to animal. Free ‘251-~-B~T~ in the bloodstream fell to less than 20% of its initial level 3 AChRs during development we labeled rat embryos hr after injection and this was defined as the time of with ‘%I-a-BuTx. Pregnant females were anesthetized with ether, the horns of the uterus were externalized, labeling. Rats were killed various times after labeling and each embryo was injected im with 0.5-1.0 pg of 1251- and the soleus muscle was removed. a-BuTx in a volume of 3-5 ~1 using a lo-p1 Hamilton Preparation of single jibers. Labeled muscles were syringe. We are indebted to Dr. A. J. Harris for dempinned to Sylgard-lined dishes, washed with saline, and onstrating this technique of intrauterine injection fixed in 0.8% glutaraldehyde and 2% formaldehyde in to us. 150 mM NaCl, 3 mM CaC12, 30 mM Hepes buffer at 4°C At appropriate times after injection the mothers overnight. A patch of muscle 5 mm square was dissected were anesthetized and three or four embryos were re- from the central portion of the diaphragm. For ectopic moved. The embryos were weighed and the diaphragms endplates, a segment of the soleus under the foreign dissected and processed for autoradiography as de- nerve and devoid of the original endplates was used. To scribed below. Postanatal data were obtained by re- obtain single fibers, fixed muscle segments were homoving diaphragms from pups. mogenized in lo-15 ml of distilled water in a VirTis In these experiments it was important that free 1251- homogenizer at setting 30-50 for several seconds and a-BuTx be rapidly removed so that relabeling of the then at setting 20 for lo-30 sec. This procedure usually receptors would not occur during the period of meabroke the muscle into long (200- to 2000-pm) single fisurement. To determine the rate of toxin removal we bers (Fig. 1). This method is more rapid and efficient injected litters at embryonic day (ED) 16 or ED 20. Six than the customary method of teasing out indivdual hours after the injection, the radioactivity in bulk car- muscle fibers (Bevan and Steinbach, 1977; Burden, cass muscle had fallen to 10% of the original level and 1977a). by 24 hr it had declined to less than 4% of the original Autoradiographv. Gelatin-coated slides containing a value. Over 70% of the remaining radioactivity was in few drops of a fiber suspension were air dried, dipped the form of toxin-receptor complex since, after soluin a mixture of two parts Kodak NTB-2 emulsion to one bilization in detergent, it was retained by DEAE filters part 3% glycerol at 4O”C, and exposed at 4°C for 8 hr and was precipitated by an antiserum to rat AChRs. to 10 days. The slides were developed for 2 min at room synapses are more accessible to experimental manipulation, we have also examined the turnover properties of AChRs during the formation of ectopic synapses in rat soleus muscle. Our results show that in both cases AChRs in newly formed clusters turn over rapidly; within 5 days, however, their turnover time changes to that of adult junctional receptors. Furthermore, we show that the continued presence of the nerve is necessary to effect this change in stability of clustered AChRs at ectopic endplates.
labilization
FIG. 1. Photomicrograph of a slide containing muscle fibers from a rat diaphragm (ED 21) prepared for autoradiography. Bar = 1 mm.
temperature (20-22°C) in a Kodak D-19 developer made up in 1% glycerol, fixed, dried, and mounted with Permount (Fisher Chemical Co.). Silver grains were counted under darkfield illumination using a Zeiss microscope with 10X eyepieces and a 40X Neofluar objective. A drawing tube was used to trace an area of muscle fiber onto graph paper which had been calibrated using a stage micrometer. It was assumed that only radioactivity on the surface of the muscle in contact with emulsion generated grains. Grain densities over unlabeled fibers or over extrajunctional regions of labeled normal adult muscles were not significantly above background grain densities. Background grain densities, measured near each muscle fiber, were not more than 10% of the grain density within clusters. No corrections were made for curvature of the fibers since only clusters that were viewed en face were counted. For extraCalculation of half-times for turner. junctional regions, the grain density was calculated by counting the number of silver grains in at least 1000 l.cm2 of each fiber surface and subtracting the background grain density. Eight fibers were analyzed per muscle and one to four muscles were examined for each time point. The mean density was plotted on semilogarithmic coordinates as a function of the time after labeling, and the half-life was determined from the slope. For clustered receptors, the number of grains per cluster was counted on four to eight fibers in each muscle and corrected for background grain density. Clusters were readily identified as areas whose grain density after subtraction of background exceeded that of surrounding areas by approximately an order of magnitude. Identification and quantitation of grains in clusters by two independent observers yielded values differing by less than 10%. Further, determination of cluster size at ectopic synapses using rhodamine-labeled a-bungarotoxin gave sizes similar to those obtained by autoradiography (Weinberg et al., 1981b). The total number of grains per cluster rather than density
of ACh
249
Receptors
was used to determine changes in the number of labeled AChRs in the cluster because significant changes in the area occupied by labeled AChRs occur following a pulse label (Weinberg, et aZ., 1981a). For ectopic synapses the mean number of grains per cluster was plotted as a function of the time after labeling and the half-time for turnover obtained as above. For synapses in developing muscle, one litter was used for each experiment. Because it was not feasible to remove embryos on a daily basis without endangering the mother, groups of two to four embryos were taken from each litter at two time points, 48-72 hr apart. In each embryo the number of grains per cluster was determined for five to eight fibers from the diaphragm. The SEM of the grains per cluster in each animal was about 10% of the mean value. The mean half-life for turnover within the litter was determined by taking the mean of the half-times calculated for each muscle taken at an early time paired with each muscle taken at a late time, assuming that the loss of radioactivity was exponential. The SEM of these values (shown in Fig. 4) represents the variability among diaphragms in a litter. An alternative method, in which all fibers from animals taken at the same time were pooled, and the mean and SEM of these values used to compute the mean and SEM of half-lives for turnover gave quantitatively similar results (data not shown). Additional methods. In some control experiments slides were stained for acetylcholinesterase through the developed emulsion for 15-30 min at room temperature (Karnovsky and Roots, 1964). Binding of ‘%I-a-BuTxAChR complexes to DEAE filters and immunoprecipitation were performed as described previously (Brockes and Hall, 197533; Weinberg and Hall, 1979). RESULTS
Developing
Muscles
We determined the apparent turnover time of AChRs in developing rat diaphragms by labeling embryos with 1251-~-B~T~ and using autoradiography to measure the loss of radioactivity from receptor clusters and from extrajunctional regions of muscle fibers. In extrajunctional regions radioactive toxin was lost rapidly from all muscles. The results of an experiment in which embryos were injected on ED 21 and the density of grains in extrajunctional regions of muscle fibers was measured 1, 2, and 3 days later are shown in Fig. 2. The grain density decreased exponentially with a half-time of 24 hr. In other experiments we determined grain density at only two times and calculated a halftime assuming that the loss of toxin was exponential. The half-times of toxin loss from extrajunctional re-
250
0.4L
1 0
I 48 Time after labeling
I 96 (hr)
FIG. 2. Extrajunctional grain density on developing muscle fibers from rat diaphragm as a function of time after labeling with ‘251-(uBuTx. The litter was injected in utero on ED 21 and the first point taken 24 hr later (at birth). Density was measured on 16-24 fibers from two or three diaphragms at each time point. Points represent mean grain density + SEM.
gions of embryonic muscle did not change between ED 18-22. This half-time of 24 + 2 hr (mean + SEM; n = 5) is in the same range as the values we obtained from denervated adult diaphragm (17 f 2 hr) and soleus (25 f 8 hr) muscles. These values also fall within the range of estimates of the turnover time for extrajunctional receptors made by other methods in denervated adult muscles (Berg and Hall, 1975; Chang and Huang, 1975) and in myotubes in cell culture (Devreotes and Fambrough, 1975). In diaphragms, clusters of AChRs first appeared at ED 15-16 (Bevan and Steinbach, 1977; Braithwaite and Harris, 1979). Although the grain clusters observed in young fibers were more diffuse than the sharply defined, dense clusters observed later, clustered receptors were always easily distinguished from those in extrajunctional regions (see Fig. 3). In general, only one cluster was seen per fiber; however, in fibers from young
muscles (ED 18 or earlier) a group of two to four small clusters was occasionally seen (see also Bennett and Pettigrew, 1974; Bevan and Steinbach, 1977) and these were excluded from turnover measurements. Over 80% of the receptor clusters at ED 16, and over 90% in older embryos, coincided with sites of acetylcholinesterase staining. Since acetylcholinesterase characterizes sites of neuromuscular transmission (Bennett and Pettigrew, 1974) we conclude that the clustered AChRs are at neuromuscular junctions. As illustrated by the autoradiographs shown in Fig. 3, ‘251-a-BuTx was lost rapidly from clusters of AChRs at the earliest times examined, but within 4 days the toxin was lost much more slowly. To characterize the rate of loss during development, we measured grain densities in clusters at two times after labeling and from these calculated apparent half-times of turnover of AChRs in clusters. For convenience, we assumed that toxin loss occurred with a single exponential process, although this may not be the case at intermediate times. The results of autoradiographic measurements of such apparent turnover times of clustered AChRs during development are summarized in Fig. 4. On ED 17 and 18, toxin was lost from clusters with a half-time of 3235 hr, which is similar to that obtained for extrajunctional receptors. By ED 21, the half-time for toxin loss exceeded 145 hr in four out of five litters; these values were not distinguishable from those obtained for junctional AChRs in adult diaphragm. Although these measurements cannot distinguish between a change in turnover rate for all clustered receptors and a change in the proportion of slowly and rapidly turning over receptors within clusters, it is clear that the AChRs at endplates in embryonic rat muscle undergo a marked increase in turnover time between ED 18 and 21, whereas extrajunctional receptor turnover times remain low throughout development.
FIG. 3. Dark-field photomicrographs of autoradiographs of muscle fibers from developing rat diaphragms. Grain clusters on fibers from litter-mates injected with ‘z51-a-BuTx on ED 17 and prepared (a) 1 day or (b) 3 days later, or from littermates injected on ED 20 and prepared (c) 2 days later (at birth) or (d) 4 days later. Bar = 20 pm. The single fibers shown in each micrograph are oriented horizontally and are approximately the same width as the micrograph.
REINESS
EJR
ED 17
ED IS
ED 19
ED 20
21
21
ED 21
AND WEINBERG
Birth
PD 8
Adult JR
AGE
FIG. 4. Half-lives for turnover of AChRs clustered at developing endplates in rat diaphragm. Each bar represents results from a separate litter. Lower axis gives age of animals at initial time point. Shown for comparison are half-lives measured for embryonic extrajunctional receptors (EJR) and junctional receptors of S&day postnatal (PD 8) or of adult rats. At each time point, a total of lo-20 clusters from diaphragms of two to four animals were analyzed except for the ED 1’7, ED 19, and one of the ED 20 litters where only a single animal (5-8 clusters) was available at the second time point. Bar represents SEM. The method used to determine turnover times in these experiments could not distinguish values in excess of 150 hr (see Materials and Methods).
Ectopic Endplates Cutting the original nerve to a muscle enables a foreign nerve previously implanted in an endplate-free zone to make new endplates (Elsberg, 1917). We used these ectopic endplates in adult muscle as a second system in which to study the change in AChR turnover time that occurs during synaptogenesis. AChRs were labeled by injection of a sublethal dose of 1251-a-BuTx that bound about 90% of the receptors in the rat soleus muscle. Muscles were removed at various times after injection and loss of radioactivity was determined by counting grains per cluster in autoradiographs of single fibers. In control experiments on normal soleus muscles the half-time for toxin loss at adult endplates was 10 f 1.5 days, which is similar to values obtained in soleus muscles (Linden and Fambrough, 1979) or rat diaphragms (Reiness et aZ., 1978) maintained in organ culture, or by autoradiography of adult diaphragm (Fig. 4). In agreement with earlier results (Berg and Hall, 1975; Chang and Huang, 1975; Linden and Fambrough, 1979) the extrajunctional AChRs that appeared after denervation turned over much more rapidly with a half-time around 1 day (25 f 8 hr).
Stabilization
251
of ACh Receptors
In muscles receiving ectopic innervation, clusters were first seen 2 days after denervation and their size and grain density increased gradually over the next few weeks. Such clusters were substantially larger than those seen by Ko et al. (1977) in denervated rat soleus muscles (Weinberg et aZ., 1981b), and were not seen in the distal nerve-free segment of ectopically innervated muscles nor in denervated soleus muscles lacking an implanted foreign nerve. The turnover time of receptors in these clusters was determined by plotting the number of grains per ectopic endplate vs time after labeling. The results of these experiments are shown in Fig. 5. Two days after denervation, toxin was lost exponentially with a half-time of 24 f 5 hr. At later times toxin was lost exponentially with longer half-times (7.5 -+ 1.5 to 10 f 1.5 days for muscles labeled 6,1’7, and 40 days after denervation). Thus the rate of toxin loss from clustered AChRs at ectopic endplates changed between 2 and 6 days after denervation from a rapid value characteristic of extrajunctional receptors to a slow value characteristic of junctional receptors. In order to determine if the change in rate of toxin loss requires the presence of the nerve, we cut the for-
’ O”
BLIP
17 days
6 days
4 days
2 days
Time after
labeling
(hr)
FIG. 5. Loss of ‘=I-cr-BuTx from newly formed ectopic endplates on rat soleus muscles. Ectopic innervation was produced by cutting the original soleus nerve 2-3 weeks after implanting a foreign nerve on an endplate-free region of the muscle. AChRs were labeled in tivo at ages ranging from 2 to 40 days after cutting the original nerve. Toxin bound to eetopic endplates was determined by counting the grains per cluster on autoradiographs of single muscle fibers. The values obtained were corrected for the extrajunctional grain density and normalized to an exposure time of 1 week with a specific activity of 100 cpm/fmole. The number of grains per cluster is plotted as a function of the time after the muscle was labeled. Each point represents the mean & SEM of three to eight (usually four to six) endplates.
DEVELOPMENTALBIOLOGY
252
eign nerve 3 days after denervating the soleus. At this time clusters have formed underneath the ectopic nerve but still consist primarily of rapidly turning over receptors. Three days later we labeled the muscles with ‘%I-cu-BuTx. Clusters were still seen at the site of ectopic innervation in these muscles which shows that maintenance of newly formed clusters does not require the presence of the foreign nerve. The number of AChRs in these clusters was unchanged from the number when the foreign nerve was cut, but the clusters were about 30% larger and more diffuse than those seen before the foreign nerve had been cut. The loss of toxin from these denervated ectopic endplates is shown in Fig. 6. Toxin was lost exponentially with a half-time of 42 + 14 hr. Thus toxin was lost more rapidly from denervated ectopic endplates than from those in which the foreign nerve remained intact (t112 = 7.5 + 1.5 days).
DISCUSSION
We have found that ‘251-a-BuTx is rapidly lost from newly formed synapses both during normal development and during ectopic innervation of adult muscle (tl,z g 1 day). Within 5 days of cluster formation, however, the toxin is lost, as from adult endplates, at a much slower rate (tl,z = 6-10 days). Previous work has shown that ‘=I-a-BuTx binds specifically to AChRs in skeletal muscle (Chang and Lee, 1963; Miledi and Potter, 1971; Berg et al., 1972) and that at least some of the loss of radioactivity from the muscle reflects internalization and degradation of the toxin-receptor complex (Berg and Hall, 1975; Devreotes and Fambrough, 1975; Libby et al., 1980). Radioactivity is lost from muscle both by turnover of the AChR-‘%I-cw-BuTx complex and by dissociation 50
5Ll 0
I 90
48 Time after labeling
(hr)
FIG. 6. Loss of r&I-a-BuTx from denervated ectopic endplates. Ectopic endplates were denervated by resecting the foreign nerve 3 days after the original nerve had been cut. Three days later (6 days after cutting the original nerve) AChRs were labeled with ‘%I-a-BuTx. Toxin bound to the denervated ectopic endplates was determined as in Fig. 5. Each point represents the mean + SEM of four to six endplates.
VOLUME
84, 1981
of the toxin from the receptor. Since the half-time for dissociation of lzT-~-BuTx from AChR of adult muscles in organ culture is about 2 weeks (Reiness et al., 1978; Linden and Fambrough, 1979), this process does not contribute appreciably to loss of toxin from rapidly turning over receptors (t1,2 zz5 days), but may constitute a substantial fraction of toxin loss from receptors which turn over more slowly. Thus our measurements of toxin loss by autoradiography are likely to underestimate the turnover time for more stable receptors. Diffusion of labeled AChRs out of clusters could increase the apparent rate of turnover, and might account for the initially rapid rate of toxin loss. Our results do not allow us to distinguish the contributions of diffusion and degradation to the rate of toxin loss.. While no data are available about the diffusion coefficient of AChRs in developing endplates, AChRs which cluster on uninnervated cultured rat myotubes are not freely diffusible (Axelrod et al., 1976). If the same is true in viva, diffusion of AChRs away from the clusters would not account for the initially rapid rate of loss. Accumulation of toxin by diffusion of labeled receptors into a cluster would decrease the apparent rate of loss of toxin. Thus the change in turnover that we describe might be due to accumulation of receptors by diffusion. We followed the calculation of Edwards and Frisch (1976) for the accumulation of receptors at an endplate. Using our measurements of cluster size, extrajunctional grain density, and turnover time, and Axelrod et uZ.‘s, (1976) measurement of 6 X 10-u cm21 set for the diffusion constant of nonclustered receptors on cultured rat myotubes, we found that less than 10% of the total label in a cluster would be added by diffusion during 24 hr. For a cluster of rapidly turning over receptors this would increase the apparent turnover time by less than 5 hr. Thus accumulation of AChRs by diffusion could not account for the increase in turnover time that we observed at new endplates. We found that AChRs in clusters at new synapses in rat muscles initially have a short half-life in the membrane that is comparable to that of extrajunctional AChRs. The rapid loss of AChRs could reflect the disappearance of the clusters themselves; however, clusters at newly formed nerve-muscle synapses in vitro persist (Frank and Fischbach, 1979). Thus the rapid loss probably reflects turnover of AChRs within the clusters. Clustering of AChRs and their metabolic stabilization are therefore independent processes in rat muscle indicating that the two processes have different mechanisms. Similar results were obtained in developing chick muscle by Burden (1977a). Clustered receptors with short half-times have also been found at long-term denervated adult endplates (Brett and Younkin, 1979; Loring and Salpeter, 1980).
REINESS AND WEINBERG
Burden (1977b) found in chicken posterior latissimus dorsi muscle that more than 2 l/2 weeks elapsed between the formation of clusters and the change in receptor turnover time. In rats, in contrast, the change to a long turnover time occurs a few days after clustering at an endplate. In rat embryos the turnover time increased to adult values between ED 17 and ED 21. This is consistent with earlier results which suggested that slowly turning over AChRs are present in neonatal (ED 22) rat diaphragms (Berg and Hall, 19’75; Steinbach et al., 1979). A similar time course was seen at ectopic endplates where most of the change in turnover time occurred between the third and the sixth days after cutting the original nerve. We showed that the continued presence of the nerve is required for the metabolic stabilization of AChRs and, as at mature synapses (Frank et al., 1975a), the nerve is not required for the maintenance of receptor clusters. The mechanism by which this stabilization takes place is not clear. The rapidly turning over receptors may either be replaced by or converted to the more slowly turning over form. Bloch and Steinbach (1979) have reported that clusters of AChRs at developing neuromuscular contacts can initially be readily dispersed by removal of Ca2+ but that receptors at older endplates are resistant to such treatment. This resistance to dispersal within the membrane may correspond to the metabolic stabilization described in this paper. Several studies indicate that the mean channel open time of AChRs at motor endplates changes later during development than the change in metabolic stability that we have described (Sakmann and Brenner, 1978; Fischbath and Schuetze, 1980). The relationship of these changes in physiological properties during development to changes in the molecular form of the AChR is not clear. The time course of the development of molecular properties such as differences in isoelectric point (Brockes and Hall, 1975c) and in antigenic determinants (Almon and Appel, 1975; Weinberg and Hall, 1979) remains to be established; however, since the changes in physiological properties follow different time courses, a single molecular alteration could not account for all of them. The results at ectopic endplates parallel those at developing embryonic endplates. In both cases clusters of rapidly turning over receptors are formed, and within 5 days the receptors are metabolically stable. Ectopic endplates offer particular advantages for the study of neuromuscular synapse formation since they mature relatively synchronously and are readily accessible to experimental manipulation. In summary, there are at least three distinct phases in the appearance of mature, junctional properties of
Stabilization
of ACh Receptors
253
AChRs during the formation of new endplates. First, AChRs become clustered at sites of nerve-muscle contact. During the subsequent 4-5 days their turnover time increases at least 5- to lo-fold. Finally, l-2 weeks later, the mean channel open time changes during development to that characteristic of adult AChRs (Sakmann and Brenner, 1978; Fischbach and Schuetze, 1980). We thank Dr. Zach W. Hall, in whose laboratory at the University of California, San Francisco, these experiments were conducted, for his advice and encouragement, and for helping to develop the technique for single fiber preparation. We also thank Miu Lam for skillful technical assistance and Liz Neville and Veronica Oliva for assistance in preparation of the manuscript. This work was supported by postdoctoral fellowships from the Muscular Dystrophy Association (MDA) and the National Institutes of Health (NIH) to C.G.R. a predoctoral fellowship from the National Science Foundation to C.B.W., and grants from MDA and NIH to Z. W. Hall.
REFERENCES ALMON, R. R., and APPEL, S. H. (1975). Interaction of myasthenic serum globulin with the acetylcholine receptor. Biochim. Biophys. Acta 393,66-W. ANDERSON, M. J., COHEN, M. W., and ZORYCHTA, E. (1977). Effects of innervation on the distribution of acetylcholine receptors on cultured muscle cells. J. Physiol. 268, 731-756. ANGULO Y GONZALEZ, A. W. (1932). The prenatal growth of the albino rat. Anat. Rec. 52,117-138. AXELROD, D., RAVDIN, P., KAPPEL, D. E., SCHLESSINGER, J., WEBB, W. W., ELSON, E. L., and PODLESKI, T. (1976). Lateral motion of fluorescently labeled acetylcholine receptors in membranes of developing muscle fibers. Proc. Nat. Acad Sci. USA 73,4594-4598. AXELSSON, J., and THESLEFF, S. (1959). A study of supersensitivity in mammalian muscle. J. Physiol. 147, 178-193. BENNETT, M. R., and PETTIGREW, A. G. (1974). The formation of synapses in striated muscle during development. J. Physiol. 241.515545. BERG, D. K., KELLY, R. B., SARGENT, P. B., WILLIAMSON, P., and HALL, Z. W. (1972). Binding of a-bungarotoxin to acetylcholine receptors in mammalian muscle. Proc. Nat. Acad. Sci. U&l 69,147-151. BERG, D. K., and HALL, Z. W. (1975). Loss of a-bungarotoxin from junctional and extrajunctional acetylcholine receptors in rat diaphragm muscle in viva and in organ culture. J. Physiol. 252, 771789. 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. (1979). The effect of Ca++-deprivation on accumulations of acetylcholine receptor at the developing neuromuscular junction. Sot. Neurosci. Abstr. 5,476. BRAITHWAITE, A. W., and HARRIS, A. J. (1979). Neural influence on acetylcholine receptor clusters during embryonic development of skeletal muscles. Nature (London) 279, 549-551. BRETT, R. S., and YOUNKIN, S. G. (1979). Accelerated degradation of junctional a-bungarotoxin-acetylcholine receptor complexes in denervated rat diaphragm. Sot. Neurosci. Abstr. 5, 765. BROCKES, J. P., and HALL, Z. W. (1975a). Synthesis of acetylcholine receptor by denervated rat diaphragm muscle. Proc. Nat. Acad. Sci. USA 72,1368-1372. BROCKES, J. P., and HALL, Z. W. (1975b). Acetylcholine receptors in
254
DEVELOPMENTAL BIOLOC:Y
normal and denervated rat muscle. I. Purification and interaction with [‘Z51]-a-bungarotoxin. Biochemistry 14, 2092-2099. BROCKES, J. P., and HALL, Z. W. (1975c). Acetylcholine receptors in normal and denervated rat muscle. II. Comparison of junctional and extrajunctional receptors. Biochemistry 14,2100-2106. BURDEN, S. (1977a). Development of the neuromuscular junction in the chick embryo: the number, distribution, and stability of acetylcholine receptors. Develop. Biol. 57.317-329. BURDEN, S. (1977b). Acetyleholine receptors at the neuromuscular junction: developmental change in receptor turnover. Develop. Biol. 61, 79-85. CHANG, C. C., and HUANG, M. C. (1975). Turnover of junctional and extrajunctional acetylcholine receptors of the rat diaphragm. Nature (London) 253,643-644. CHANG, C. C., and LEE, C. Y. (1963). Isolation of neurotoxins from the venom of Bungarus multicinctus and their modes of neuromuscular blocking action. Arch. Int. Pharmacodyn. 144,241~257. 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 of foetal and new-born rat muscle fibres. J. Physiol. 162, 393-408. EDWARDS, C., and FRISCH, H. L. (1976). A model for the localization of acetylcholine receptors at the muscle endplate. J. Neurobiol. 7, 377-381. 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. FISCHBACH, G. D., and SCHUETZE, S. M. (1980). A postnatal decrease in acetylcholine channel open time at rat endplates. J. Physiol. 303, 125-137. FRANK, E., and FISCHBACH, G. D. (1979). Early events in neuromuscular junction formation in vitro. J. Cell. BioZ. 83.143-158. FRANK, E., GAUTVIK, K., and SOMMERSCHILD, H. (1975a). Cholinergic receptors at denervated mammalian motor end-plates. Acta Physiol. Stand. 95, 66-76. FRANK, E., JANSEN, J. K. S., LIMO, T., and WESTGAARD, R. H. (1975b). The interaction between foreign and original motor nerves innervating the soleus muscle of rats. J. Physiol. 247, 725-743. HARTZELL, H. C., and FAMBROUGH, D. M. (1972). Acetylcholine receptors: Distribution and extrajunctional density in rat diaphragm after denervation correlated with aeetylcholine sensitivity. J. Gen. Physiol. 60, 248-262. HEINEMANN, S., MERLIE, J., and LINDSTROM, J. (1978). Modulation of acetylcholine receptor in rat diaphragm by anti-receptor sera. Nature (London) 274, 65-68. KARNOVSKY, M. F., and ROOTS, L. (1964). A “direct-coloring” thio-
VOLUME 84, 1981 choline method for cholinesterase. J. Histochem. Cytochem. 12,219-
221. 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. LEE, C. Y., TSENG, L. F., and CHIU, T. H. (1967). Influence of denervation on localization of neurotoxins from elapid venoms in rat diaphragm. Nature fLondcm) 215, 1177-1178. LIBBY, P., BURSZTAJN, S., and GOLDBERG, A. L. (1980). Degradation of the acetylcholine receptor in cultured muscle cells: Selective inhibitors and the fate of undegraded receptors. Cell 19.481-491. LINDEN, D. C., and FAMBROUGH, D. M. (1979). Biosynthesis and degradation of acetylcholine receptors in rat skeletal muscles. Effects of electrical stimulation. Neuroscience 4, 527-538. 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. LORING, R. H., and SALPETER, M. M. (1980). Denervation increases turnover rate of junctional acetylcholine receptors. Proc. Nat. Acad. Sci. USA 77, 2293-2297. MILEDI, R. (1960). The acetylcholine sensitivity of frog muscle fibres after complete or partial denervation. J. Physiol. 151, l-23. MILEDI, R., and POTTER, L. T. (1971). Acetylcholine receptors in muscle fibres. Nature (London) 233,599~603. REINESS, C. G., WEINBERG, C. B., and HALL, Z. W. (1978). Antibody to acetylcholine receptor increases degradation of junctional and extrajunctional receptors in adult muscle. Nature (London) 274,6870. SAKMANN, B., and BRENNER, H. R. (1978). Change in synaptic channel gating during neuromuscular development. Nature (London) 276, 401-402.
STEINBACH, J. H., MERLIE, J., HEINEMANN, S., and BLOCH, R. (1979). Degradation of junctional acetylcholine receptors by developing rat skeletal muscle. Proc. Nat. Acad. Sci. USA 76, 3547-3551. WEINBERG, C. B., and HALL, Z. W. (1979). Antibodies from patients with myasthenia gravis recognize determinants unique to extrajunctional acetylcholine receptors. Proc. Nat. Acad. Sci. USA 76, 504-508. WEINBERG, C. B., REINESS, C. G., AND HALL, E. W. (1981a). TOPOgraphical segregation of old and new acetylcholine receptors at developing eetopic endplates in adult rat muscle. J. Cell Biol. 88, 215-218. WEINBERG, C. B., SANES, J. R., AND HALL, R. W. (1981b). Formation of neuromuscular junctions in adult rats: accumulation of acetylcholine receptors, acetylcholinesterase, and components of synaptic basal lamina. Develop. Biol. 84,255-266.