Ultrastructural evidence of continued reorganization at the aging (11 У26 months) rat soleus neuromuscular junction.код для вставкиСкачать
THE ANATOMICAL RECORD 207:399-415 (1983) Ultrastructural Evidence of Continued Reorganization at the Aging (11-26 Months) Rat Soleus Neuromuscular Junction CONSTANCE ANN CARDASIS Department of Anatomy, University of Massachusetts Medical School, Worcester, MA 01605 ABSTRACT Ultrastructural remodeling, with evidence of focal deafferentation and reinnervation, occurs within normal young adult rat soleus neuromuscular junctions (Cardasis and Padykula, 1981). This may be related to normal variations in function. Recognition of this plasticity provides a basis for analysis of aging changes in junctional ultrastructure. Thirty soleus junctions were studied between 11 and 26 months of life. In these junctions, compared to younger ones (3-5 months) synaptic sites with the conventional ultrastructure become increasingly sparse. There is a n increase in extent and frequency of exposed junctional folds, of intervention of Schwann cell cytoplasm between axon and junctional folds, and of numbers of lysosomes in all cytoplasmic profiles. Often primary clefts are shallow or missing, and secondary folds are widened and contain collagen. Features limited largely to these older junctions include highly pleomorphic myonuclei, deeply invaginated by myofibrils, and a n increase in cellular profiles between basal lamina and sarcolemma. The identity of these profiles is unknown. At other locations within many of the same endplates, small intact terminals are associated with larger expanses of junctional folds, and several small terminals occur within the same primary cleft. Such terminals frequently contain dense-cored vesicles. These observations suggest continuation of some terminal axonal regeneration. Thus, the ultrastructure of these aging neuromuscular junctions reveals the same degenerative and regenerative events suggested by the ultrastructure of younger junctions, but suggests a shift in the balance between them. The neuromuscular junction (NMJ), as the 1981; Cardasis and Padykula, 1981) and amfinal common pathway of motor systems, is a phibian (Letinsky et al., 1976; Wernig et al., key site for analysis of nerve-muscle interac- 1980) NMJs suggest continuous structural tion during aging and senescence. The senes- and functional remodeling. This may be recent NMJ has been reported to exhibit defects lated to normal variation in functional dein synaptic transmission (Frolkis et al., 19761, mands during growth or transient changes diminished ability to sustain neuromuscular in workload. Consideration of the phenometransmission (Smith, 1979)and alterations in non of junctional reorganization during morphology which suggest decreased axo- young adulthood is critical for appropriate plasmic flow (Gutmann and Hanzlikova, analysis and interpretation of aging NMJ 1976), and/or focal denervation (Gutmann structure. Studies of whole mounts of rat soand Hanzlikova, 1976; Fujisawa, 1976; Pes- leus endplates, stained by heavy metal imtronk et al., 1980; Cardasis, 1981). However, pregnation and reacted for cholinesterase the literature suggests that there is both spe- activity, reveal a n increase in endplate size cies and muscle variability in the develop- and in the number of axonal branches with ment of senile alterations (reviewed in age in the SD rat (3 and 28 months) (Fagg et Spencer and Ochoa, 1981).Most studies treat al., 1981) and in the Wistar rat (2, 10, and 18 “aging” as a distinct entity in contrast to months) (Pestronk et al., 1980), whereas a (young) adulthood. However, adulthood decrease in endplate length and in the comshould not be regarded as a static endpoint plexity of axonal terminals occurs between of differentiation. Morphologic studies of adult mammalian (Barker and Ip, 1966; Tuffery, 1971; Pestronk et al., 1980; Fagg et al., Received December 20, 1982; accepted July 6, 1983. 0 1983 ALAN R. LISS, INC. 400 C.A. CARDASIS TABLE 1. Body and soleus muscle weight (gms) and soleus cross-sectional area (mrn’) of individual male rats ICI~-CIrl:~ORS-ISII))RR) a ~ 11-26 d months Age (months) 11 11 14 14 15 15 18 18 19 19 22 22 23 23 26 26 ~~ % R soleus wt. Body wt. Right soleus wt. 532.6 553.6 630.5 661.6 596.4 583.5 822.4 672.5 565.3 642.9 668.5 749.2 613.0 669.4 728.5 692.3 0.27 0.22 0.33 0.26 0.25 0.25 0.30 0.30 0.22 0.28 0.30 0.28 0.20 0.17 0.27 0.22 Left soleus wt. 0.31 0.26 0.33 0.33 0.30 0.25 0.25 0.23 body wt. 0.051 0.040 0.052 0.039 0.041 0.043 0.036 0.045 0.043 0.038 0.045 0.037 0.032 0.025 0.038 0.032 Crosssectional area 15.4 12.33 13.45 10.27 8.33 18 and 28 months in the Wistar rat (Pestronk Schwann cell cytoplasm. The basal lamina of et al., 1980). However, there is little infor- Schwann cells is typically folded or multilaymation concerning aging junctional ultra- ered, possibly as a result of Schwann cell structure. mobility. A high degree of synthetic activity The adult rat soleus muscle, frequently of the junctional sarcoplasm is suggested by studied as a n example of a predominantly the usual junctional organelles such as polyslow twitch muscle, has been described in somes, rough endoplasmic reticulum, and both morphological and physiological inves- Golgi, and the consistent finding of “myofitigations (Gutmann and Hanzlikova, 1965, brillar segments” and membranous triads 1976; Vyskocil and Gutmann, 1971)as one of oriented in various planes. Cumulatively, the preferential sites of the aging process. these findings at young adult rat soleus juncDuring young adulthood, the postural soleus tions suggest remodeling that includes axmuscle is subject to increasing workload by onal sprouting and withdrawal and regencontinuous body growth. The soleus in- erative anabolic signs in the junctional sarcreases in weight proportionally to body coplasm. Such synaptic plasticity may be reweight. This is accompanied by myofiber hy- lated to the response of this postural muscle pertrophy and conversion of motor units to a to continuous body growth andlor alterations slow type (Kugelberg, 1976). Body growth in activity of laboratory rats. levels off beyond 9 months of age (Table l), These structural reorganizations a t normal and workload may even be reduced as older adult soleus junctions provide a basis for rats generally become less active (Cohen et analysis of age changes in junctional ultraal., 1978). In the senescent rat soleus, atro- structure. During the second year of life sophy and loss of myofibers result in a decline leus workload is no longer increasing, in in the size of individual motor units (Gut- contrast to the period of 3-9 months when mann et al., 1968) and a decrease in the slow workload increases. The present study is a twitch fiber population (Caccia et al., 1979). comparison of the ultrastructure of young Soleus junctions of the young adult SD rat adult (3-5 months; group I) and older adult (3-5 months) reveal several ultrastructural (11-26 months; group 11) soleus NMJs. Evisigns of reorganization (Cardasis and Padyk- dence will be sought to clarify the relationula, 1981).Focal sites of denervation are sug- ship between the reorganization during adultgested by the presence of exposed junctional hood and degeneration during senescence. folds occurring adjacent to typical intact synMATERIALS AND METHODS aptic associations. In addition, some axonal Body and Soleus Muscle Weights terminals, presumably in the process of either withdrawal or reinnervation are sepBody and soleus muscle weights were dearated from myofibers by intervening termined for 16 male rats (C6-Crl: COBS- AGING RAT SOLEUS NEUROMUSCULAR JUNCTIONS (SD)BR) (Charles River Breeders). Body weights for two rats of each age 11, 14, 15, 18, 19, 22, 23, and 26 months were obtained prior to chloroform anesthesia. The right and left soleus muscles were dissected out in their entirety, pinned a t resting length to a preweighed waxed petri dish, and weighed. The data on body weight of these aged rats were combined with body weight determinations of younger rats obtained previously in this laboratory (Cardasis and Padykula, 1981).This data was analyzed statistically to obtain the curve of best fit. The growth curve was used to determine a t what age the rate of growth slows, thus changing the increasing workload placed on the soleus muscle. The Pearson regression coefficient and its probability were derived for soleus muscle versus body weight and for the soleus muscle weight expressed as percentage body weight versus age on rats aged 9-26 months. These data were used to determine whether or not the linear relationship that exists between these variables during young adulthood (Cardasis and Padykula, 1981)is altered. 401 endplate, composite electron micrographs at low magnification as well as selected areas at higher magnifications were obtained with a JEOL JEM-100s electron microscope. Approximately 30 soleus motor endplates were examined in this manner. The frequency with which certain ultrastructural features were viewed in ultrathin sections was quantitated in young adult (3-5 month; group and older adult (11-26 months; group 11) junctions. This percentage was derived from all welloriented junctions in which photographic composites along the entire length were available. The data for young adult junctions (3-5 months) were obtained by reexamination of 23 junctions employed in a n earlier study (Cardasis and Padykula, 1981). The statistical significance of the differences between groups I and I1 was analyzed by corrected Chi square and/or Fisher exact tests. Measurements of Area of Cross Section at Area of the Soleus Muscle The left soleus muscle and its nerve from five of the rats were exposed and fixed in situ with 2.5% glutaraldehydel2% paraformaldehyde in 0.1 M cacodylate buffer (Karnovsky, 1965). The soleus nerve was removed and processed separately. The left soleus muscle was removed, and fixation continued by immersion for 2 hr. The tissue was washed and stored in 0.1 M cacodylate-sucrose buffer (pH 7.4). The left soleus was sectioned across its belly, dehydrated, and embedded in methacrylate. Cross sections (2 km) were cut on a JB-4 microtome and stained with hematoxylin and eosin. Composite photomicrographs of the entire muscle were taken a t low magnification with a Zeiss photomicroscope for the determination of muscle cross-sectional area. Area measurements were performed with the aid of digitizing pad linked to a Digital Equipment Corporation Computer Model PDP-11-40 according to the planimeter program which gives a running total of trapezoid areas swept out by the cursor (100 coordinateslsec 0.005" accuracy). Ultrastructural Analysis of Motor Endplates The ultrastructure of soleus motor endplates was investigated in ten of the rats used to obtain soleus and body weights. The rats were aged 11, 15, 19,23, and 26 months (2 rats per age). The pinned muscles from the right limb were fixed immediately following weighing by immersion in 6.25% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 2 hr. The muscles were washed and stored in 0.1 M cacodylate-sucrose buffer (pH 7.4). The junctional region was identified by reacting whole muscles for nonspecific esterase, employing alpha-napthyl acetate as substrate (Gomori, 1950) as described previously (Cardasis and Padykula, 1981). Blocks of tissue containing the motor endplates were postfixed in 1%OsO4 in veronal acetate buffer (pH 7.41, stained en bloc with 0.5% uranyl acetate, dehydrated, and embedded in Epon 812. Semithin plastic sections (0.75 pm) were cut from longitudinally oriented blocks and RESULTS stained with toluidine blue to locate motor Body and Soleus Muscle Weight endplates. Ultrathin sections were prepared from these blocks, mounted on 100-mesh slot Figure 1illustrates that the rate of growth grids, and stained with uranyl acetate and of Sprague Dawley male rats is rapid during lead hydroxide (Karnovsky, 1961). young adulthood and slows at approximately Because of the sampling problem in elec- 9 months of age. Thus, beyond 9 months of tron microscopy, composite micrographs that age the postural soleus muscle is not subinclude the entire length of a junction in jected to the same continuous increase in section were used in analysis. For each motor workload that is imposed by rising body C.A. CARDASIS 402 eoDy WEIGMT OF MALE SPRAGUE OAWLEY RATS AT VARIOUS AGES BOO1 . too 0 0 . 2 . 4 . 6 . B . 10 . 12 . 14 . 16 . I8 , 20 . 22 . 24 . 26 Age lmonlhsl Fig. 1. Graph of body weight in individual male CDCrl:COBS-(SD)BR rats from 1 month to old age. Body weight increases rapidly during young adulthood (2-9 months). Beyond 9 months of age there is relatively little weight gain. The best fitting curve that describes the growth has the equation: Weight = 256.7 i 148 X In (age). The R2 of 0.75 indicates that 75% of the variation is accounted for by this curve. weight during young adulthood. The strong positive linear correlation that exists between increasing body and soleus muscle weights prior to 9 months of age (Pearson R = 0.962; P = < 0.0001) (Cardasis and Padykula, 1981) is not present between 9 and 26 months of age (Pearson R = 0.322; P = 0.104) (Table 1).The average contribution of soleus weight to body weight between 2 and 9 months of age is 0.043%. The Pearson R (0.287) for soleus wet weight expressed as a percentage of body weight versus age in young adult rats (2-9 months) is not significant. In contrast to this positive relationship in young growing rats, rats beyond 9 months of age show a significant negative correlation (Pearson R = -0.662; P = 0.002). It is probable that, rather than a linear relationship, the data would more closely fit a curve that declined rapidly with advancing age. The data suggest that there is a decline in soleus weight, accompanied by a decrease in crosssectional area by 23 months of age (Table 1). However, a larger sample size is necessary to substantiate this. The ultrastructure of soleus motor endplates was examined during the period of 11 to 26 months of age when the relationship between workload and increasing muscle weight is altered. with synaptic vesicles and mitochondria opposed to secondary synaptic folds of the muscle. Ultrastructural evidence of degeneration and regeneration as well as normal synaptic contact areas occur side by side within individual aging soleus endplates. However, analysis of composite electron micrographs of longitudinally oriented endplates reveals that, with advancing age, normal contact areas become increasingly sparse. This decreases the effective area of synaptic contact within junctions. A decrease in the area of synaptic contact is indicated by the following ultrastructural features: 1) Regions of exposed junctional folds a t each aging junction are more extensive P i g . 2A) than in young adult rats. 2) Often the primary clefts are shallow or entirely missing (Fig. 2A-C). 3) Remodeling and Degeneration at Soleus Motor Endplates Synaptic contact areas with the conventional ultrastructure persist within junctions throughout 11 to 26 months of age. These “normal” areas include axonal terminals Fig. 2. A) Eleven months. Electron microscopic composite of a longitudinally sectioned soleus motor endplate. Note the extensive area of exposed secondary folds (arrows).Only two small axon terminals (AT) are associated with small sites of this region of secondary folds. The two “satellite cells” (SC) exhibit variations in the cytoplasmic density. Schwann cell (Sch). Perineural epithelium (PN). ~4,000. Higher magnifications of the 2 axon terminals are included in 7B,C. B) A small axon terminal (AT) is associated with the openings of two junctional folds, one of which contains cross sections of collagen fibrils (C). The remainder of the folds are exposed. Preterminal axon (A). Junctional folds (JF). Schwann cell cytoplasm (Sch). ~ 1 7 , 2 0 0 C) . A higher magnification electron micrograph of an axon terminal (AT). This terminal lies in a shallow primary cleft and is associated with the openings of 3 secondary folds (JF)in this plane of section. In addition to the typical clear synaptic vesicles, the axon contains a dense-cored vesicle (DV) and coated vesicle (CV). Schwann cell cytoplasm (Sch). Folded or redundant Schwann cell basal lamina (BL). x 19,300. AGING RAT SOLEUS NEUROMUSCULAR JUNCTIONS 403 404 C.A. CARDASIS TABLE 2. Comparison of young adult (group I) and aging (group II) soleus junctional ultrastructure' Group Terminals Axonal terminal totally ~ysosomes~ with dense- Pleomorphic Intrabasal isolated from Exposed folds by Schwann Axon Junctional cored postjunctional lamina cellular folds' Schwann cell cell terminal sarcoplasm vesicles' myonuclei profiles' 14 (3-5 months) N = 23 I1 (11-26 months) N = 17 61 9 70 26 56 4 4 40 100 29 94 53 88 67 29 82 'This comparison is expressed as the percentage of NMJs observed in photographic composites derived from ultrathin sections that include these ultrastructural features. 'Statistically significant differences between groups I and I1 (see text). 3Lysosomes include dense bodies and secondary lysosomes and residual bodies identified on the basis of structure alone. Multivesicular bodies of the junctional sarcoplasm are not included. 4Data obtained by quantification of ultrastructural features in junctions employed in an earlier study (Cardasis and Padykula, 1981). Some secondary folds are widened and contain collagen fibrils (Fig. 2B). 4)Axonal terminals are often separated from junctional folds by Schwann cell cytoplasm (Fig. 3). The percentage of junctions with exposed folds during young adulthood (Cardasis and Padykula, 1981)was revised upward. The prior study included only extensive regions of exposed folds, whereas here junctions with any number of exposed folds were included. Nevertheless, during aging the percentage of junctions viewed in ultrathin sections which include exposed folds and axonal terminals enveloped by Schwann cells is increased above adult values (Table 2). The increase in the proportion of NMJs with exposed folds in the older group is statistically significant (Fisher exact test, P = 0.0059). Axonal terminals (Figs. 3,4) and Schwann cells (Fig. 5) occasionally contain large pleomorphic membrane-limited structures, a feature not observed at young adult endplates. Whether these structures are totally or partially enveloped by membrane is not established. The pleomorphic bodies within axonal terminals contain structures that might, based on their size and shape, be remnants of synaptic vesicles and mitochondria. With the exception of this distinctive structure, terminal Schwann cells a t both young adult and aging endplates share common structural features associated with highly active cells. Their prominent Golgi zone is commonly associated with lysosomes and centrioles. There is a small increase in structures resembling lysosomes in the Schwann cells of the older rats (group 11). However, without acid phosphatase localization the possibility exists that some of these represent other organelles (Fig. 6). Although sign of focal denervation is the most prominent ultrastructural feature a t every aging soleus motor endplate, the following evidence also suggests that some terminal axonal regeneration may occur within these endplates. 1) Small, ultrastructurally normal axon terminals are associated with inappropriately large expanses of mature junctional folds which sometimes exhibit signs of prior denervation (lack of primary cleft, collagen fibrils located within secondary folds) (Figs. 2B,C, 6). 2) Several small axon terminals occur within the same primary cleft and are often isolated from one another by Schwann cell cytoplasm (Figs. 7, 8). Such axon terminals usually contain dense-cored vesicles as well as the usual clear synaptic vesicles (Figs. 2C, 7,8). Dense-cored vesicles are far more common in axonal terminals a t aging junctions, whereas they are Fig. 3. Twenty-three months. A pleomorphic membrane bound body (*I, which encloses structures that might represent remnants of vesicles, occurs within an axonal terminal. This ultrastructural feature has not been observed at young adult endplates and may be related to degeneration. The terminal is completely surrounded by Schwann cell cytoplasm (SCH) in this section, isolating it from the junctional folds (JF).Synaptic and coated vesicles (V).Mitochondria (MI. x 15,300. Fig. 4. Twenty-three months. Similar pleomorphic structures (*) are located within this soleus axonal terminal, along with the usual synaptic vesicles (SV) and mitochondria 04). Schwann cell cytoplasm (SCH). Junctional folds (JF).x 19,000, AGING RAT SOLEUS NEUROMUSCULAR JUNCTIONS 405 406 C.A. CARDASIS AGING RAT SOLEUS NEUROMUSCULAR JUNCTIONS quite rare during young adulthood (Table 2). The increase in the proportion of NMJs with dense cored vesicles is highly statistically significant (P = 0.0004).Furthermore, within aging junctions dense-cored vesicles are rarely observed in terminals that appear to be degenerating, i.e., terminals containing autolytic vesicles and/or isolated from junctional folds by Schwann cell cytoplasm. Axonal terminals containing round or elongated coated vesicles are more frequently encountered during aging. Fifty-two percent of axonal terminal profiles from aging rats include such vesicles as compared to 33% of adult axonal terminals. Also, in each axonal profile the number of coated vesicles increased from one to two with age. Postsynaptic Structures The sarcoplasmic organelles of both young and aging adult junctions include mitochondria, ribosomes, rough endoplasmic reticulum, microtubules, and microfilaments. Multivesicular bodies are frequently observed during all ages (22% of adults and 25% of aging junctions). However, other secondary lysosomes are more numerous at aging junctions (Fig. 7; Table 2). Coated vesicles, apparently arising from secondary folds, and other small vesicles are numerous in the junctional sarcoplasm intervening between folds at aging junctions. “Myofibrillar segments” oriented in various axes occur in both the young adult (Cardasis and Padykula, 1981) and aging soleus junctions. However, in the aging junctional sarcoplasm, these myofibrillar segments are closely associated with highly pleomorphic myonuclei (Figs. 9-12). Invaginations of these junctional myonuclei create cytoplasmic pockets Fig. 5. Twenty-three months. A Schwann cell (SCH) contains a pleomorphic structure (*I similar to those observed within axonal terminals (see Figs. 3,4). The usual cytoplasmic organelles of both young adult and aging Schwann cells, such as rough endoplasmic reticulum (RER), mitochondria (M), and a prominent Golgi zone (G), indicate a high degree of synthetic activity. Redundant basal lamina (BL) and lysosomes (L)are also a consistent feature of adult and aging Schwann cells. Axonal terminal (A). Junctional folds (JF).x7,OOO. Fig. 6. Eleven months. Small axon terminals (A) are apposed by a Schwann cell (SCH) on one side and the junctional folds (JF)on the other. These may represent regenerating axons. Collagen is located within a junctional fold. The Schwann cell containing a Golgi zone (G) associated with a centriole (C) and membrane-bound granules of undetermined significance. Junctional myonucleus (N). x 11,000, 407 that consistently contain “myofibrillar segments,” triads, and ribosomes (Fig. 10, 11). Ten of the 61 junctional myonuclei observed were of this pleomorphic type (16%),whereas during young adulthood such myonuclei were observed only once in 43 junctional myonuclei (2%). Also in the older group more NMJs included pleomorphic myonuclei in thin section (Table 2). Electron micrographs raise the possibility of a n association of the I band filaments and the outer membrane of the nuclear envelope. At other sites the outer nuclear envelope is studded with ribosomes. Pleomorphic myonuclei are invaginated to varying degrees (Fig. 9,101. The invaginations may become so pronounced as to suggest destruction (Fig. 11). “Satellite cells” are frequently associated with both adult and aging junctional sarcoplasm (Fig. 2). Unlike satellite cells located elsewhere along fiber, basal lamina intermittently intervenes between these satellite cells and the myofiber. There is a large increase in the number of intrabasal lamina cellular profiles in aging junctions (P = 0.0159) (Table 2). These profiles do not always include a nucleus (Fig. 121, and sometimes the cytoplasm is very similar to that of the junctional sarcoplasm. It is possible that these represent satellite cells or partitions of junctional sarcoplasm. However, the identity of these profiles is not established. Ultrastructural Alteration of Aging Myofi bers During the second year of life, in contrast to the first year, muscle weight no longer increases. However, muscle atrophy is not clearly detectable before 23 months of age (Table 1).Ultrastructural alteration is distinctly evident in the myofibers by 15 months of age. The most prevalent alteration is the presence of accumulations of enlarged mitochondria in predominantly subsarcolemmal locations (Fig. 13A-C). Often the cristae cannot be discerned within part or all of the mitochondria section (Fig. 13B,C). Thus, the organization of the inner membrane and matrix becomes distinctly different. The following structural alterations are also evident in some aging myofibers (Fig. 13A-C): 1) Extracted areas most likely occupied by glycogen are common, particularly at sites containing enlarged mitochondria; 2) Lysoma1 structures are frequently observed a t the poles of aging myonuclei; 3) Polysomes are closely associated with filaments; 4)Ac- 408 C.A. CARDASIS AGING RAT SOLEUS NEUROMUSCULAR JUNCTIONS 409 reversion to a smaller, less complex axonal terminal branching pattern seen in whole mounts during late aging (28 months) of the Wistar rat soleus (Pestronk et al., 1980). DISCUSSION In contrast t o the “normal” structure of These results indicate that continuous exposed folds during the growth phase, durmorphological reorganization occurs at both ing aging there is often loss of primary clefts growing young adult (3-5 months; group I) and widening of the exposed secondary folds. and older adult (11-26 months; group II) These widened folds characteristically conjunctions. Group I1 includes both rats whose tain collagen fibrils. Thus, regions of exposed rate of growth has slowed and older rats ex- folds a t aging junctions may have been dehibiting senescent changes. Within individ- nervated for a longer period of time than the ual group I1 junctions, ultrastructural signs exposed folds of young adult junctions. Exof degeneration and regeneration occur side perimental denervation studies demonstrate by side with normal areas of synaptic con- that primary cleft structure is lost early (Pultact. However, quantitative summarization liam and April, 19791, whereas secondary of the data (Table 2) shows that in these folds may persist for more than 5 months aging adults, morphological signs of degen- (Miledi and Slater, 1968). By late aging (23 eration are more prominent, relative to signs and 26 months), exposed folds without priof regeneration, than in growing adults. The mary clefts are commonly located at considpercentage of NMJs that include the ultra- erable distances from the few remaining structural features quantitated in Table 2 is axonal terminals (Fig. 9). This alteration in higher in all cases in group 11. However, the postsynaptic architecture may be related to differences are not all statistically signifi- the decrease in ChE (cholinesterase) activity cant. This probably reflects the limitations of demonstrated in whole mounts of aging mussample size and/or the wide range of ages in cle (Gutmann and HanzlikoCa, 19651, where it was often limited to only a “thin rim” of group 11. The most universal sign that suggests de- the endplate. The decline in the amount of normal syngeneration is a decrease in the effective area of synaptic contact. Regions of exposed syn- aptic contact area within aging junctions may aptic folds become increasingly more exten- at some point result in a decline in the trophic sive with age and are present within 100%of interaction of nerve and muscle and in diffithe aging junctions examined, rather than in culties in transmission (Frolkis et al., 1976; the 33% reported for young adult soleusjunc- Smith, 1979). Such a decline would account tions (Cardasis and Padykula, 1981).Similar for the disruption in the ultastructural orgafindings of exposed folds coexisting with nor- nization of myofibers (15 months) and their mal terminals have been reported in nine out subsequent atrophy. Physiologic studies of of ten aging rat median thigh muscles ex- mouse soleus NMJs demonstrate an increase amined by EM (Fujisawa, 1976). In the pres- in the EPP and the safety factor during agent investigation, although normal synaptic ing (Robbins and Kelly, 1981).These authors contact areas are observed at all ages, they speculate that increased turnover of synaptic become increasingly rare with advancing age. vesicles compensates for the observed deThis ultrastructural observation supports the crease in the number of synaptic vesicles (Fahim and Robbins, 1981, 1982). At aging rat soleus terminals, the increase in coated vesicles and in pleomorphic vesicles may be reFig. 7. Twenty three months. Several small axon terlated to more rapid synaptic vesicle turnover minals (A) partially isolated from each other by Schwann during aging. cell cytoplasm (arrows) are located within one primary cleft. Two terminals contain dense-cored vesicles (DV). Studies of the aging neuromuscular system Coated vesicles (CV) are located in sarcoplasm between in laboratory animals may be complicated by junctional folds (JF).X 17,600. disease arising concurrently with aging. For example, guinea pigs have been reported to Fig. 8. Eleven months. Two axonal terminals (A) within one primary cleft are isolated from each other by develop a hind limb neuropathy which is Schwann cell cytoplasm (arrows). Note the presence of a thought to arise from ambulation in wiredense-cored vesicle (DV) in each terminal as well as clear bottomed cages (Fullerton and Gilliatt, 1967). synaptic vesicles (SV). Sarcoplasm contains ribosomes However, the findings of ultrastructural flux (R) and a multivesicular body (MVB). Junctional folds (JF).Schwann cell lysosomes (Lys). X 15,200. reported for the adult rat soleus are identical cumulations of elements of smooth endoplasmic reticulum and T tubule system occur in the sarcoplasm. 410 C.A. CARDASIS AGING RAT SOLEUS NEUROMUSCULAR JUNCTIONS in rats raised solely in either wire mesh or solid-bottomedcages (unpublished results). These degenerative aspects of aging junctional structure may, in part, arise from a decrease in the rat of axoplasmic transport reported during aging (McMartin and O’Connor, 1979). Such a decrease has been implicated in impaired neuromuscular transmission, neurotrophic action, and axonal regeneration (Drahota and Gutmann, 1961). Certain alterations at the aging motor endplate are not manifest during young adulthood. Aging junctional myonuclei are commonly invaginated so highly as to form numerous cytoplasmic pockets, which regularly contain “myofibrillar segments” as defined by Cardasis and Padykula (1981). A possible factor in the development of this nuclear pleomorphism may be the local contraction of the “myofibrillar segments” exerting force on the nuclear envelope. An association between actin filaments and the outer membrane of the nucler envelope of extrajuncctional myonuclei has been reported (Franke and Schinko, 1969). However, a subsequent study of isolated myonuclei demonstrates that the invaginated state is not only a result of contractile elements (Franke, 1970). The mechanism by which the highly invaginated aging junctional myonuclei are formed remains to be established, along with their significance. These heterochromatic, pleomorphic myonuclei most likely are not active in protein synthesis. In fact, the invaginations may become so extensive (Fig. 12) as to suggest that nuclear invagination may represent a stage in the destruction of the postmitotic myonuclei. Disturbances of nerve-muscle interaction during aging might first be manifested in the nuclei of the postjunctional sarcoplasm prior to the senile atrophy of the myofiber. Fig. 9. Twenty-three months. Composite electron micrograph of soleus endplate. It differs from the young adult junction in the following respects: 1)Exposed junctional folds (arrows) are present at a considerable distance from axonal terminals (A) and even extend beyond the field in this micrograph. 2) The postjunctional myonuclei include two highly pleomorphic nuclei (N1 and N2) associated with “myofibrillar segments”(*). 3) Schwann cells (SCH) exhibit numerous lysosomes (L).The cellular structure located near the axon terminals may represent attempts at regeneration of new myofibers 04). Myelinated (M-SN) and unmyelinated (SN) branch of soleus nerve. Capillary (C). X4,lOO. 411 “Myofibrillar segments” are quite extensive during the growth phase of young adulthood (Cardasis and Padykula, 1981), but during aging they are largely confined to the region of pleomorphic myonuclei. Their apparent decrease during aging, particularly late aging, when myofiber size is either stable or undergoing atrophy lends support to the hypothesis that they are related to myofiber growth rather than to degeneration. The occurrence of both partial denervation and reinnervation at aging endplates is suggested by AgIChE (silver/cholinesterase) staining methods of whole mounts at the aging Wistar rat soleus (Pes tronk et al., 1980). In addition to the ultra structural features suggesting focal denervation, analysis of aging soleus junctions also suggest that terminal axonal sprouting not only continues but may even increase during early aging. Often, between 11 and 15 months, axonal profiles suggesting regeneration are the only ones present in composite longitudinal sections (Fig. 2). Perhaps this occurs in response to partial denervation induced by normal withdrawal of axonal terminals, as experimentally induced partial denervation of a muscle results in increased sprouting of remaining axons at normally innervated endplates (Brown and Ironton, 1978;Rotskenker, 1978). Sprouting and reinnervation of aging soleus endplates are indicated by certain ultrastructural features that are similar to those observed during experimental regeneration of adult endplates (Lullmann-Rauch, 1971). Small healthy axonal terminals are sometimes associated with larger expanses of junction folds, which often exhibit the signs of previous denervation noted above (Fig. 2). Commonly, several small axonal terminals, isolated from one another by Schwann cell cytoplasm,occupy the same primary cleft. The presence of densecored vesicles and the usual clear synaptic vesicles is a consistent finding in such terminals. These ultrastructural features are reminiscent of developing NMJs during the period of polyneuronal innervation (PNI)(Korneliussenand Jansen, 1976).It is possible that during this active degeneration and sprouting in aging terminals, some PNI may occur, possibly as a preliminary stage in normal collateral sprouting. There is some morphologic evidence that suggests (Pestronk et al., 1980)that PNI may occur in very old Wistar rat soleus. 412 C.A. CARDASIS AGING RAT SOLEUS NEUROMUSCULAR JUNCTIONS An increase in the number of axonal terminals with aging is supported by heavy metal stains of various muscles (Tuffery, 1971; Rosenheimer and Smith, 1981; Pestronk et al., 1980; Fagg et al., 1981). However, the shapes of some of these terminals suggest that they are not forming functional synaptic contact (Tuffery, 1971).It is possible that they are withdrawn with further aging. In 28-month-oldrat diaphragm, a decline was noted in the percentage of axons exhibiting both terminal degeneration and regeneration (Rosenheimer and Smith, 1981). In the Wistar rat soleus, fewer branch points and a decrease in overall endplate size were reported at 27 months (Pestronk et al., 1980). Similarly, a decrease in ultrastructural evidence of sprouting occurs during the latter stages of aging (23 and 26 months). Axonal regeneration in response to experimental nerve section is reduced with advancing age (Drahota and Gutmann, 1961). This decline in regenerative capacity could result in axonal sprouting being inadequate to compensate for the normal withdrawal of terminals, and it might account for the decrease in synaptic contact area during aging. Another significant postsynaptic change seen during aging is difficult to relate to either degeneration or regeneration. There is a n increase in the number of cellular profiles located within the muscle basal lamina Vable 2). Some include a nucleus (Figs. 2A, 12) while others are only cytoplasm (Fig. 12). A similar finding has been reported following experimental denervation (Miledi and Sla- 413 ter, 1968). Thus, this increase during aging may be associated with the decreased normal synaptic contact area within aging junctions. The identity of these profiles remains to be established. They may originate from several sources, including fragmentation of the myofiber, an increase in the number or change in the shape of satellite cells, or they may originate from migration of Schwann cells to a subbasal lamina position. An important consideration is that the interaction of nerve and muscle and the structure of the NMJ are intimately associated with activity levels during postnatal development (Benoit and Changeux, 1975; Riley, 1978; O’Brien et al., 1978) and adulthood (Holland and Brown, 1980; Brown et al., 1980). The gradual transitions of NMJ ultrastructure described here may not necessarily represent inevitable results of aging per se. It is likely that both intrinsic (neurotrophic) and extrinsic (exercise, nutrition) factors affect the morphology of aging endplates (Gutmann and Hanzlikova, 1972) and vice versa. However, the present study has defined the ultrastructural plasticity of the NMJ of a particular, well-studied muscle during young adulthood and aging. Thus, it provides a baseline upon which to investigate molecular events and to test the effects of various experimental manipulations on the aging of the NMJ. Furthermore, studies of the plasticity of this well-studied synapse may provide a useful model for the understanding of certain aspects of synaptic plasticity in the central nervous system. ACKNOWLEDGMENTS Fig. 10. Fifteen months. An invagination (arrow) of this pleomorphic junctional myonucleus (N) forms a cytoplasmic pocket containing myofibrillar segments (*I. Ribosomes (R) are associated with the outer membrane of the nuclear envelope. x 11,000. Fig. 11. Eighteen months. This structure in the junctional sarcoplasm may represent nuclear segments that are degenerating. Note the more normal appearance of the junctional myonucIeus (N). ~8,200. Fig. 12 Twenty-three months. Two closely associated intrabasal lamina profiles are located in the perijunctional region. One (1) includes a nucleus (N), while the other (2) is only a thin cytoplasmic process of greater electron density. The cytoplasm of the nucleated profile contains ribosomes, short cisternae of rough endoplasmic reticulum, and mitochondria and resembles the underlying muscle sarcoplasm (S). X 11,000. This investigation was supported by research grants from the Muscular Dystrophy Association of America and National Science Foundation BNS-8207829. The author wishes to express her appreciation to medical student Donna LaFontaine for assistance in the quantitative aspects of this study, Scrantz Lersch and Christopher Hebert for providing photographic assistance, and Elsie Larson for the preparation of the manuscript. The author would like to thank Drs. Merrill K. Wolf and Susan Billings-Gagliardi for their encouragement and for critical reading of the manuscript. Discussions with Dr. Helen A. Padykula provided a valuable foundation for this work. Fig. 13. Myofibers themselves begin to exhibit die, tinct signs of ultrastructural alterations by 15 months of age. A) Twenty-three months. The sarcoplasm contains lysosomal-like structures (Lys), polysomes (R) associated with single filaments (F), enlarged mitochondria @I) and extracted areas which most likely contained glycogen. x 15,300. B) Fifteen months. 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