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Ultrastructural evidence of continued reorganization at the aging (11 У26 months) rat soleus neuromuscular junction.

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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. The sarcoplasm contains en-
larged mitochondria tM)and elements of a smooth membrane system, possibly smooth endoplasmic reticulum
(SER). ~21,000.
C) Fifteen months. Two enlarged subsarcolemmal mitochondria @I) exhibit structural alteration of the matrix and cristae. Cristae are absent
throughout one mitochondria1 profile, while the other
profile contains only a small area of cristae (C). ~ 3 8 , 5 0 0 .
AGING RAT SOLEUS NEUROMUSCULAR JUNCTIONS
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