/. Embryol. exp. Morph. 76, 37-49 (1983)
Printed in Great Britain © The Company of Biologists Limited 1983
37
Postnatal muscle fibre histochemistry in the rat
By K. W. HO 1 , W. W. HEUSNER, J. VAN HUSS AND
W. D. VAN HUSS
From the Human Energy Research Laboratory, Michigan State University
SUMMARY
Histochemical techniques were used to study the postnatal muscle fibre differentiation
patterns in the plantaris and soleus muscles of male Sprague-Dawley rats. Nine groups of
animals (n = 6/group) were killed at 1, 6, 11, 16, 21, 26, 31, 36, and 140 days of age. Serial
transverse sections of the two muscles were stained with H & E, NADH-D, and myofibrillar
ATPase with acid (pH4-35) or alkali (pH 10-4) preincubation. In each of the age groups, all
available fibres across the muscle sections were classified. Obtained data show that fibre types
are basically undifferentiated at birth in both muscles.
In the plantaris muscle there are about 99 % type IIA and less than 1 % type Ifibresat 6 days
of age. Type IIB fibres can be identified at 11 days of age. There are increases in the percentages of type Ifibres(from 0-7 % to 3-5 %) and type IIBfibres(from 1-1 % to 6-5 %) between
6 and 11 days and between 16 and 21 days respectively. By 36 days of age the relative numbers
of type IIA, IIB, and type I fibres in the plantaris are approximately 80 %, 14 %, and 6 %,
respectively. A gradual change infibre-typecomposition continues until it becomes 47 % for
type IIA, 43 % for type IIB, and 10 % for type I at 140 days of age.
In the soleus muscle there are approximately 73 % type IIA and 26 % type I fibres at both
6 and 11 days of age. However, type IIA fibres decrease to 44 % and type Ifibresincrease to
56 % at 16 days of age. This rapid shift infibrecomposition continues up to 31 days of age when
the distribution becomes 25 % for type IIA and 74 % for type I fibres. Thereafter, the differentiation rate is much slower. At 140 days of age, there are 17 % type IIA and 83 % type
I fibres in the soleus muscle.
The results of this study show that the fibre populations in the plantaris and soleus muscles
of the rat undergo a postnatal differentiation process. In both muscles the adult fibre population is established by 140 days of age. Although relatively rapid increases of type I and type
IIB fibres occur in the plantaris during the second and third weeks of life, differentiation in
that muscle appears to be an essentially continuous process. There is a notable shift in the fibre
composition of the soleus muscle during the second postnatal week. Differences between the
patterns of differentiation in the two muscles are apparent.
INTRODUCTION
It is well known that skeletal muscles are functionally organized into motor
units and that the muscle fibres in a given motor unit are homogeneous in terms
of their metabolic properties, their recruitment patterns in performing various
activities, and their potential work capacities (Burke & Edgerton, 1975). Many
useful systems of fibre-type classification have evolved according to the morphological, physiological, biochemical, and histochemical characteristics of different
1
Author's address: Human Energy Research Laboratory, Department of Health and Physical Education, Michigan State University, East Lansing, Michigan 48824, U.S.A.
38
K. W. HO AND OTHERS
muscle fibres, but the widely accepted nomenclature of Brooke & Kaiser (1970)
consisting of type I, IIA, IIB, and IIC was adopted for use in this investigation.
Numerous studies have been conducted on the properties of mature muscle.
However, the development of fibre types has not been fully understood. It is
believed that skeletal muscle fibres in the rat are basically undifferentiated at
birth (Engel & Karpati, 1968; Shafiq, Asiedu & Milhorat, 1972; Riley, 1977a)
and that the fibre populations of muscles in the adult rat are determined by a
postnatal differentiation process (Shafiq et al. 1972; Curless & Nelson, 1976;
Riley, 1977a; Haltia, Berlin, Schucht & Sourander, 1978). Shafiq et al. (1972)
reported that type I and type II fibres can be identified in the gastrocnemius and
extensor digitorum longus (fast muscles in the adult rat) and in the soleus (a slow
muscle in the adult rat) by 2-3 weeks of age. Haltia et al. (1978) found that type
IIB and type IIA fibres were evident in the extensor digitorum longus muscle at
5 days and at 15-20 days of age, respectively.
The percentages of various fibre types in a muscle are dynamic during the
period of postnatal development. In the extensor digitorum longus muscle, increases in type I fibres (from 10 % to 28 %) and type IIA fibres (from 0 % to
40 %) accompanied by decreases in type IIB fibres (from 41 % to 27 %) and in
type IIC fibres (from 49 % to 5 %) have been observed between 5 and 180 days
of age (Haltia et al. 1978). In the soleus muscle, a 10 % increase in type I fibres
and a corresponding 10 % decrease in type IIA fibres between 11 and 26 days of
age also were reported (Riley, 1977a). On the other hand, Curless & Nelson
(1976) found that the relative number of type I fibres in the soleus held constant
at about 60 % from birth to 29 days of age. The discrepancies in these studies may
be due, in part, to strain differences and to the fact that only portions of muscle
sections were used for fibre-type classification.
Because the patterns of postnatal muscle fibre differentiation are not clear at
present, this study was designed to investigate developmental changes in the
fibre-type populations of the plantaris and soleus muscles in the rat using two
traditional histochemical procedures. To avoid sampling problems associated
with the fact that fibre types are not evenly distributed across various regions of
muscle in the rat (Ariano, Armstrong & Edgerton, 1973), all available fibres in
transverse muscle sections were classified at specified times during the postnatal
development period.
MATERIALS AND METHODS
Nine groups of male albino rats (Sprague-Dawley strain, Spartan Research
Animal Inc., Haslett, Michigan) were killed at 1, 6, 11,16, 21, 26, 31, 36, and
140 days of age. There were six rats per age group. The animals were
anaesthetized by an intraperitoneal injection of sodium pentobarbital. The right
plantaris, a fast muscle in the adult rat consisting mostly of type IIA and IIB
fibres with only a small number of type I fibres, and the right soleus, a slow
Postnatal muscle fibre histochemistry in the rat
39
muscle in the adult rat consisting of a large percentage of type I fibres and a small
percentage of IIA fibres, were immediately removed and frozen in isopentane
precooled (-170°C) with liquid nitrogen.
Serial transverse sections (8/im) were cut on a microtome in a cryostat at
- 2 0 °C from the middle of each muscle belly. These muscle sections then were
stained with haematoxylin-eosin (H&E), reduced nicotinamide adenine
dinucleotide diaphorase (NADH-D) (Novikoff, Shin & Drucker, 1961), and
myofibrillar adenosine triphosphatase (myo-ATPase) (Padykula & Herman,
1955) with acid (pH4-35) or alkali (pH10-4) preincubation (Guth & Samaha,
1969).
The histochemically stained serial sections from each muscle were projected
with a Prado Universal Microprojector (Ernst Leitz Gmbh Wetzlar) in a darkroom. The staining intensity of each muscle fibre in each section was evaluated
subjectively using a rating scale of dark, medium, and light (Peter et al. 1972).
According to these ratings, each fibre was typed using the classification system
of Brooke & Kaiser (1970). Mean values of the total numbers and percentages
of different fibre types were calculated for each muscle in each age group. All
histochemical evaluations were performed blind.
RESULTS
In newborn rats, fibres from the plantaris and soleus muscles cannot be differentiated with the two histochemical procedures used. That is, the staining
intensity of NADH-D is homogeneous across each muscle section, and myoATPase reactions following acid (pH4-35) or alkali (pH10-4) preincubation
show little selective activity among the myotubes.
At 6 days of age, the staining intensity of NADH-D is uniformly dark across
all sections from the plantaris muscle. Using myo-ATPase, approximately 99 %
of the fibres stain dark with preincubation at pH 10-4, and 0-7 % of the remaining
fibres stain dark with preincubation at pH4-35. Therefore, these fibres can be
classified as type IIA and type I, respectively. Differences in stain intensity for
NADH-D start to show in the plantaris by 11 days of age. At this time type IIB
fibres become identifiable, and the relative frequency of type I fibres rises to
3-5 %. By the 16th day, the distribution of type IIA, type IIB, and type I fibres
is approximately 95 %, 1 % and 3 %, respectively. These values change to 88 %,
6-5 % and 4 % at 21 days of age and to 80 %, 14 % and 6 % at 36 days of age.
Thereafter, a gradual change in the fibre-type profile continues until the
distribution becomes 47 % for type IIA, 43 % for type IIB and 10 % for type I
fibres at 140 days of age (Fig. 1). The fibre population at this time is similar to
that previously reported for the plantaris muscle in adult rats (Ariano et al. 1973).
In the soleus muscle, the staining intensity of NADH-D is undifferentiated
until 11 days of age, but slow and fast fibres can be identified by the two myoATPase stains at 6 days of age. There are approximately 73 % type IIA and 26 %
40
K. W. HO AND OTHERS
100
Type I fibre
Typ e H A fibre
Type IIB fibre
5
90
80
70
~ 60
<u
Q.
|
50
E
40
30
20
10
0
21
26
Days of age
140
Fig. 1. Fibre type composition of the plantaris muscle as a function of age. Each
point represents the mean and standard deviation of six rats. (Unshown standard
deviations <1%.)
•
o
100
• Type I fibre
-o Type IIA fibre
90
80
|
50
40
30
20
10
0
11
16
21
26
Days of age
31
36
140
Fig. 2. Fibre type composition of the soleus muscle as a function of age. Each point
represents the mean and standard deviation of six rats.
type I fibres at both 6 and 11 days. However, the distribution changes rapidly
between 11 and 16 days. The relative frequency of type IIA fibres drops to 44 %
while that of the type I fibres rises to 55 %. This rapid shift in the fibre-type
profile continues up to 31 days of age, at which time the distribution is 25 % for
Postnatal muscle fibre histochemistry in the rat
41
Fig. 3. Temporal sequence offibre-profilechanges in the soleus muscle at selected
postnatal ages. Type I (light) and type IIA (dark)fibresare indicated (myo-ATPase
stain, pH 9-4). (A). Section from an 11-day-old animal with 26 % type Ifibresin the
soleus muscle. X250. (B). Section from a 16-day-old animal with 55 % type I fibres.
X160. (C). Section from a 26-day-old animal with 69% type I fibres. xlOO. (D).
Section from a 36-day-old animal with 73 % type I fibres, x 100.
type IIA and 74 % for type I fibres. Thereafter, the differentiation rate is much
slower. At 140 days of age there are approximately 17 % type IIA and 83 % type
I fibres in the soleus muscle (Fig. 2). This fibre population is almost identical to
Fibre type
Type I
Type IIA
Type IIB
Unclassified
Total
Type I
Type IIA
Type IIB
Unclassified
Total
Muscle
Plantaris
Soleus
16
552 ± 141
1521 ± 154
17 ± 6
2090 ± 70
17 ± 3
2169 ± 574
1558 ± 162
1057 ± 209
6±2
2602 ± 277
9±2
2702 ± 332
179 ± 56
3674 ± 1061
269 ± 122
38 ±34
4160 ± 1116
21
1499 ± 193
1193 ±158
151 ± 48
162 ±178
4470 ±1238 4667 ± 362
7±8
53 ±39
5±7
68 ±62
4644 ± 1405 4939 ± 365
11
574 ± 206
1577 ± 369"
5 ±13
4088 ± 596
29 ±34
4054 ± 576
6
6±3
2438 ± 234
8±4
2293 ± 399
1706 ± 306
580 ± 146
1673 ± 253
760 ± 68
31
98 ±41
3695 ± 485
488 ± 212
190 ± 65
4471 ± 669
26
143 ± 64
3513 ± 859
245 ± 91
59 ±36
3960 ± 974
Age (days)
104
4±2
2670 ± 216
1942 ± 180
724 ± 173
3±2
1843 ± 222
1522 ± 162
318 ± 62
229 ± 39
252 ± 16
3356 ± 841 1244 ± 100
606 ± 262 1126 ± 26
27 ±16
20 ±14
4218 ± 1047 2642 ± 125
36
Table 1. Means and standard deviations for absolute frequencies of different fibre types in the plantaris and soleus muscles at
various ages (n =6 per age group)
F
w
X
h-j
O
0
y
>-*
X
o
to
Postnatal muscle fibre histochemistry in the rat
43
that previously reported by Ariano et al. (1973) for adult rats. Fig. 3 shows a
temporal sequence of fibre profile changes in the soleus muscle at selected postnatal ages.
Table 1 gives the means and standard deviations for the absolute frequencies
of the different types of fibres observed at various ages in the two muscles. It
should be noted that the values presented may not represent the actual total
numbers of fibres in the muscles. Occasional losses of small portions of tissue
may have occurred during the histochemical preparation. Furthermore, Gollnick, Timson, Moore & Riedy (1981) recently pointed out the limitations of
using histological methods for determining absolute fibre counts in muscles.
DISCUSSION
The results of this study support the observation that traditional myo-ATPase
and NADH-D histochemical procedures are unable to differentiate muscle fibre
types in newborn rats (Engel & Karpati, 1968; Shafiq et al. 1972; Riley, 1977a;
Haltia et al. 1978).
Several investigators have claimed that a population of type IIC fibres is
present in rat muscles during early development and that these fibres may be
precursors to the later appearing fibre types (Brooke & Kaiser, 1970; Curless
& Nelson, 1976; Haltia et al. 1978). In the current study, the large number of
fibres that reacted positively to both the myo-ATPase (pH10-4) and the
NADH-D stains in the plantaris muscle at 6 days of age, although classified as
type IIA, may have included some of these transitional type IIC fibres. A
definite pattern of fibre differentiation seems to start in the plantaris after 6 days
of age which results in an essentially continuous decline in type IIA fibres and
steady increases in type I and type IIB fibres until the adult fibre composition
is attained (Fig. 1).
The differentiation pattern in the soleus muscle shows a noticeable shift in the
fibre population between 11 and 16 days of age (Fig. 2). This is in contrast to the
data of Curless & Nelson (1976) in which there was a non-significant change in
the percentage (53-65 %) of type I fibres in randomly selected fields of the soleus
muscle between 1 and 29 days of age in the same strain of rat. Likewise, Riley
(1977a) found a change of only 10 % (49-59 %) in the relative frequency of type
I fibres between 11 and 26 days of age in the soleus muscles of Long Evans rats.
Although, Riley counted all fibres in each muscle, fibre types were determined
on an average of only 395 fibres per muscle. It is well known that fibre types
usually are not uniformly distributed across the muscle in lower animals. For
example, a relatively high concentration of type IIA fibres can be found in the
area next to the medial head of the gastrocnemius in a typical transverse section
of the adult rat soleus. In the present study, all available fibres across the entire
transverse section of the muscle were classified instead of only the fibres appearing in selected areas. The discrepancy in fibre distribution between the results of
44
K. W. HO AND OTHERS
this study and those previously reported by Curless & Nelson (1976) and Riley
(1977a) may be due, in part, to this procedural difference.
Fluorescently labelled antibodies and/or electrophoretic techniques recently
have been used to identify different polymorphic forms of myofibrillar proteins
in skeletal muscles. Staining serial cross sections of adult muscles by the immunoperoxidase technique with antibodies to each of the purified proteins in
turn has shown that slow and fast components of troponin are located in type I
and type II fibres respectively and, furthermore, that a-tropomyosin is restricted
to type II fibres and jS-tropomyosin exists only in type I fibres (Dhoot, Frearson
& Perry, 1979; Dhoot & Perry, 1979). Different molecular forms of myosin also
have been identified in type I and type II fibres (Lowey & Risby, 1971; Weeds,
Hall & Spurway, 1975; Rubinstein et al. 1978). In addition, Gauthier & Lewey
(1979) have suggested that another myosin isoenzyme may exist which is
localized in the type IIA fibres. Therefore, the presence of a single polymorphic
form of each of the myofibrillar proteins in a given fibre type appears to be a
feature of adult skeletal muscle. However, this observation does not apply at all
stages of muscle development.
It has been reported that in foetal and early postnatal muscles all cells can
synthesize the fast forms of troponin. Cells that are identified as presumptive
type I fibres also can synthesize the slow forms of troponin (Dhoot & Perry,
1980). The exact forms of myosin present in immature muscle fibres are not yet
clear. Based upon cross reactivity with antibodies against both adult fast and
slow myosin, Gauthier, Lowey & Hobbs (1978) claim that slow and fast forms
of myosin coexist in all fibres during early development. Rowlerson (1979),
using only antibodies to slow adult myosin, reports that all cells in foetal and
newborn muscle contain some slow myosin; whereas, other investigators conclude that fast myosin is predominant (Chi, Rubinstein, Strahs & Holtzer, 1975;
Rubinstein & Holtzer, 1979). There is other evidence for the existence of an
embryonic form of myosin (Whalen, Butler-Browne & Gros, 1978; Hoh &
Yeoh, 1979; Whalen et al. 1979). Any failure to completely purify the myosin
that is used as an antigen and/or any failure to affinity purify the myosin
antibodies could explain, in part, the conflicting results obtained in these studies
(Rubinstein & Holtzer, 1979). In addition, Dhoot & Perry (1979) point out that
polymorphic forms of troponins and tropomyosin are single polypeptide chains
which presumably are the products of single genes. Therefore, specific
antibodies can be used to identify the form(s) of each regulatory protein present
in a given cell. The myosin molecule, however, consists of two large and four
small subunits. Each polymorphic form is the product of at least three structural
genes. Localization studies using antibodies against only part of the molecule,
therefore, cannot provide definitive information about the exact nature of the
whole myosin molecule.
Although the exact forms of myosin present in immature muscle cells are still
to be determined, contractile proteins are known to be synthesized around the
Postnatal muscle fibre histochemistry in the rat
45
time when myoblasts fuse to yield myotubes (Devlin & Emerson, 1978; Whalen
et al. 1978). Regulatory proteins also can be identified in foetal muscle during
early gestation (Dhoot & Perry, 1980) when the motor nerves have very little,
if any, differential activity (Diamond & Miledi, 1962). Furthermore, there is
much evidence now for the synthesis of these myofibrillar proteins in cultured
embryonic tissue where neural innervation is absent (Whalen etal. 1978; Rubinstein & Holtzer, 1979; Whalen et al. 1979). Therefore, the initial expression of
genes responsible for the synthesis of various myofibrillar proteins can be considered to be an intrinsically programmed event in developing muscle. However
it is also hypothesized that the activity of these genes is under some kind of
coordinated control during postnatal development (Rubinstein & Holtzer, 1979;
Dhoot & Perry, 1980).
The fact is well established that both the contractile properties and the forms
of myosin in adult muscle can be altered by the type of innervation provided.
For example, either cross-reinnervation with a 'slow' nerve or prolonged lowfrequency stimulation can convert a fast muscle into a slow muscle with transformed myosin properties (Buller, Eccles & Eccles, 19606; Salmons & Vrbova,
1969; Weeds, Trentham, Kean & Buller, 1974; Salmons & Sreter, 1976).
Transformation of the type of troponin complex also results from crossreinnervation of adult muscle (Amphlett et al. 1975).
A similar nerve-muscle interaction is seen during early development. The
normal differentiation of contractile activity of the muscle can be altered by
denervation or cross-reinnervation (Buller et al. 1960a; Gordon & Vrbova,
1975). For example, it is known that fibre-type differentiation does not occur
following neonatal denervation in rat skeletal muscle. Histochemical and
electron microscopic results have shown that fibre types are well differentiated
in normal gastrocnemius, extensor digitorum longus, and soleus muscles at 2-3
weeks of age, but differentiation is not seen in denervated muscles (Shafiq et al.
1972).
Even during the course of normal muscle development, there is indirect
evidence to suggest the existence of a neural influence on muscle fibre differentiation. Fibres in neonatal rat muscles are polyneuronally innervated.
During the first 2-3 weeks of postnatal life, however, there is a marked transition
from polyneuronal to unineuronal innervation of muscle fibres (Redfern, 1970;
Benoit & Changeux, 1975; Brown, Jansen & Van Essen, 1976; Korneliussen &
Jansen, 1976). Riley (19776) reported that the percentage of rat soleus muscle
histologically innervated by two or more terminal axonal branches decreases
from 73 % to 9 % between 11 and 15 days of age. While studying correlated
morphological and physiological changes during the elimination of terminals,
O'Brien, Ostberg & Vrbora (1978) observed that polyneuronal innervation can
no longer be detected electrophysiologically in the rat soleus at 15 days of age
although some muscle fibres still show histological polyneuronal innervation.
Rosenthal & Taraskevich (1977) also reported a rapid loss of polyneuronal
46
K. W. HO AND OTHERS
innervation in the rat diaphragm, a fast muscle, between 8 and 14 days of age with
a roughly linear decline in the average number of axons innervating each muscle
fibre from 2 • 4 to 1 • 1. These data suggest that by 2 weeks of age most muscle fibres
in the rat have only a single functional junction and that extra terminals about
to be retracted are in fact non-functional. It should be noted that these rapid
changes in innervation pattern coincide with the most intense growth period of
the spinal anterior horn cells in rats (Haltia, 1970). Of further interest is the fact
that several investigators have observed the synthesis of fibre-type-specific
regulatory and contractile proteins correlates well in time with the elimination
of polyneuronal innervation (Gauthier etal. 1978; Dhoot & Perry, 1979; Dhoot
& Perry, 1980). For example, Dhoot & Perry (1980) used Sprague-Dawley rats
to show that although all cells in gestation synthesize fast forms of troponins, cells
identified as presumptive type I fibres also can synthesize slow forms. In these
presumptive type I fibres, the amount of fast troponins progressively decreases
while the slow forms steadily increase after birth. By 10-11 days, the fast and
slow forms of troponin components are completely segregated into separate
muscle fibres.
If the synthesis of specific forms of myofibrillar proteins in a given muscle cell
is under coordinated control during postnatal muscle development, one might
expect that a marked alteration in the innervation pattern should be reflected by
a somewhat parallel response in the fibre population of the muscle. The results
of this study confirm such a relationship. It was shown that approximately 51 %
of the eventual type I fibre population appears in the rat soleus, a slow muscle,
between 11 and 16 days of age. This time span corresponds exactly to the period
of maximal polyneuronal elimination observed by Riley (1977b) and O'Brien et
at. (1978) in the same muscle. As for the plantaris, approximately 30 % of the
adult type I fibre population appears between 6 and 11 days, a period which
corresponds closely with that reported by Rosenthal & Taraskevich (1977) for
rapid loss of polyneuronal innervation in the rat diaphragm, another fast muscle.
Such concurrent changes suggest that a relationship exists between the
polyneuronal elimination pattern and the muscle fibre differentiation pattern,
but it would be premature to ascribe cause and effect to the relationship.
The role played by muscular activity in the coordinated control of postnatal
muscle fibre differentiation is intriguing but even more tenuous. As early as 1970
Redfern suggested that activity might be an important factor in polyneuronal
elimination because the period of maximum elimination coincides with a general
increase in the activity of the animal. This theory was supported by the observation that the elimination process is delayed when muscle activity is reduced either
by tenotomy (Benoit & Changeux, 1975) or by nerve block (Thompson, Kuffler
& Jansen, 1979) and is accelerated by low-frequency electrical stimulation (8 Hz)
of the motor nerve for 4-6 h per day (O'Brien et al. 1978). Of course, since the
relationship between polyneuronal elimination and fibre-type differentiation has
not been defined fully, the demonstrated influence of muscular activity on
Postnatal muscle fibre histochemistry in the rat
47
polyneuronal elimination cannot be extrapolated to fibre-type differentiation at
this time. On the other hand, Jones (1981) did show that slow-frequency stimulation (10Hz), between 4 and 12 days of age, prevents the development of fast
contraction times in the normally fast extensor digitorum longus and tibialis
anterior muscles and yields a very large proportion of histochemically identified
slow fibres in the extensor digitorum longus. Stimulation of the normally slow
soleus muscle, however, produced inconclusive results in Jones' study.
Further evidence that muscular activity may assist in the regulation of muscle
fibre differentiation has been provided by Gutmann, Melichna, Herbrychowa &
Stichova (1976) who concluded that mechanical loading is needed for normal
differentiation to take place. A compatible observation is that an intact afferent
impulse from the muscle is essential (McArdle & Sansone, 1977).
The mechanism by which the fibre population is established in a muscle is not
clear at present. During the initial stages of muscle development, the synthesis
of various forms of myofibrillar proteins apparently is an intrinsically
programmed event. However, the importance of coordinated neural control
during postnatal growth is evident, and this neural control may be mediated by
muscular activity.
The results of this study show that the adult fibre profiles in the plantaris and
soleus muscles of the rat are developed through a postnatal differentiation
process which is completed by 140 days of age. Although relatively rapid increases in the percentages of type I and type IIB fibres occur in the plantaris
during the second and third weeks of life, the differentiation process in that
muscle follows an essentially continuous pattern. A somewhat different pattern
of differentiation is found in the soleus muscle in that a notable shift in fibre
population occurs during the second postnatal week.
REFERENCES
AMPHLETT, G. W., BROWN, M. D., PERRY, S. V., SYSKA, H. & VRBOVA, G. (1975). Cross
innervation and the regulatory protein system of rabbit soleus muscle. Nature 257,602-604.
ARIANO, M. A., ARMSTRONG, R. B. & EDGERTON, V. R. (1973). Hindlimb muscle fiber populations of five mammals. J. Histochem. Cytochem. 21, 51-55.
BENOIT, P. & CHANGEUX, J. P. (1975). Consequences of tenotomy on the evolution of multiinnervation in developing rat soleus muscle. Brain Res. 99, 354-358.
BROOKE, M. H. & KAISER, K. K. (1970). Musclefibertypes. How many and what kind? Archs
Neurol. 23, 369-379.
BROOKE, M. H., WILLIAMSON, M. D. & KAISER, K. K. (1971). Behavior of four fiber types in
developing and reinnervated muscle. Archs Neurol. 25, 360-366.
BROWN, M. C , JANSEN, J. K. S. & VAN ESSEN, D. (1976). Polyneuronal innervation of
skeletal muscle in new born rat and its elimination during maturation. /. Physiol. 261,
387-422.
BULLER, A. J., ECCLES, J. C. & ECCLES, R. M. (1960a). Differentiation of fast and slow
muscles in the cat hind limb. J. Physiol. 150, 399-416.
BULLER, A. J., ECCLES, J. C. & ECCLES, R. M. (19606). Interactions between motoneurones
and muscles in respect of the characteristic speeds of their responses. /. Physiol. 150,
417-439.
48
K. W. HO AND OTHERS
R. E. & EDGERTON, V. R. (1975). Motor unit properties and selective involvement in
movement. In Exercise and Sport Sciences Reviews, Vol. 3 (ed. J. H. Wilmore & J. Keogh),
pp. 31-81. New York: Academic Press.
CHI, J., RUBINSTEIN, N., STRAHS, K. & HOLTZER, H. (1975). Synthesis of myosin heavy and
light chains in muscle cultures. /. Cell Biol. 67, 523-537.
CURLESS, R. G. & NELSON, M. B. (1976). Developmental patterns of rat muscle histochemistry. /. Embryol. exp. Morph. 36, 355-362.
DEVLIN, R. B. & EMERSON, C. P., JR. (1978). Coordinate regulation of contractile protein
synthesis during myoblast differentiation. Cell 13, 599-611.
DHOOT, G. K., FREARSON, N. & PERRY, S. V. (1979). Polymorphic forms of Troponin T and
Troponin C and their localization in striated muscle cell types. Expl Cell Res. 122,339-350.
DHOOT, G. K. & PERRY, S. V. (1979). Distribution of polymorphic forms of troponin components and tropomyosin in skeletal muscle. Nature 278, 714-718.
DHOOT, G. K. & PERRY, S. V. (1980). The components of the troponin complex and development in skeletal muscle. Expl Cell Res. 127, 75-87.
DIAMOND, J. & MILEDI, R. (1962). A study of foetal and new-born rat muscle fibres. /.
Physiol. 162, 393-408.
ENGEL, W. K. & KARPATI, G. (1968). Impaired skeletal muscle maturation following neonatal
neuroctomy. Devi Biol. 17, 713-723.
GAUTHIER, G. F., LOWEY, S. & HOBBS, A. W. (1978). Fast and slow myosin in developing
muscle fibres. Nature 274, 25-29.
GAUTHIER, G. F. & LOWEY, S. (1979). Distribution of myosin isoenzymes among skeletal
muscle fiber types. J. Cell Biol. 81, 10-25.
GOLLNICK, P. D., TIMSON, B. F., MOORE, R. L. & RIEDY, M. (1981). Muscular enlargement
and number of fibers in skeletal muscles of rats. /. Appl. Physiol.: Respirat. Environ.
Exercise Physiol 50, 936-943.
GORDON, T. & VRBOVA, G. (1975). The influence of innervation on the differentiation of
contractile speeds of developing chick muscles. Pflugers Arch. ges. Physiol. 360,199-218.
GUTH, L. & SAMAHA, F. J. (1969). Qualitative differences between actomyosin ATPase of
slow and fast mammalian muscles. Expl Neurol. 25, 138-152.
GUTMANN, E., MELICHNA, J. & SYROVY, I. (1974). Developmental changes in contraction time,
myosin properties and fibre pattern of fast and slow skeletal muscles. Physiol. bohemoslov.
23, 19-27.
GUTMANN, E., MELICHNA, J., HERBRYCHOWA, A. & STICHOVA, J. (1976). Different changes in
contractile and histochemical properties of reinnervated slow soleus muscles of the guinea
pig. Pflugers Arch. ges. Physiol. 364, 191-194.
HALTIA, M. (1970). Postnatal development of spinal anterior horn neurones in normal and
undernourished rats. A quantitative cytochemical study. Acta physiol. scand., Suppl. 352,
1-70.
HALTIA, M., BERLIN, O., SCHUCHT, H. & SOURANDER, P. (1978). Postnatal differentiation and
growth of skeletal muscle fibres in normal and undernourished rats. /. Neurol. Sci. 36,
25-39.
HOH, J. F. Y. & YEOH, G. P. S. (1979). Rabbit skeletal myosin isoenzymes from fetal, fasttwitch and slow-twitch muscles. Nature 280, 321-323.
JONES, R. (1981). The influence of electrical activity on the development of newborn innervated rat muscles. Pflugers Arch. ges. Physiol. 391, 68-73.
KORNELIUSSEN, H. & JANSEN, J. K. S. (1976). Morphological aspects of the elimination of
polyneuronal innervation of skeletal muscle fibers in newborn rats. /. Neurocytol. 5,
591-604.
LOWEY, S. & RISBY, D. (1971). Light chains from fast and slow muscle myosins. Nature 234,
81-85.
MCARDLE, J. J. & SANSONE, F. M. (1977). Re-innervation of fast and slow twitch muscle
following nerve crush at birth. J. Physiol. 271, 567-586.
NOVIKOFF, A. B., SHIN, W. & DRUCKER, J. (1961). Mitochondrial localization of Oxidative
enzymes. Staining results with two tetrazolium salts. /. Biophys. Biochem. Cytol. 9,47-61.
O'BRIEN, R. A. D., OSTBERG, A. J. C. & VRBOVA, G. (1978). Observations on the elimination
BURKE,
Postnatal muscle fibre histochemistry in the rat
49
of polyneuronal innervation in developing mammalian skeletal muscle. /. Physiol. 282,
571-582.
PADYKULA, H. A. & HERMAN, E. (1955). The specificity of the histochemical method for
adenosinetriphosphatase. /. Histochem. Cytochem. 3,170-195.
PETER, J. B., BARNARD, R. J., EDGERTON, V. R., GILLESPIE, C. A. & STEMPEL, K. E. (1972).
Metabolic profiles of three fiber types of skeletal muscle in guinea pigs and rabbits.
Biochemistry 11, 2627-2633.
PETTE, D., VRBOVA, G. & WHALEN, R. C. (1979). Independent development of contractile
properties and myosin light chains in embryonic chick fast and slow muscle. Pfliigers Arch.
ges. Physiol. 378, 251-257.
REDFERN, P. A. (1970). Neuromuscular transmission in new-born rats. /. Physiol. 209,
701-709.
RILEY, D. A. (1977a). Multiple innervation of fiber types in the soleus muscles of postnatal
rats. Expl Neurol. 56, 400-409.
RILEY, D. A. (19776). Spontaneous elimination of nerve terminals from the endplates of
developing skeletal myofibers. Brain Res. 134, 279-285.
ROSENTHAL, J. L. & TARASKEVICH, P. S. (1977). Reduction of multiaxonal innervation at the
neuromuscular junction of the rat during development. /. Physiol. 270, 299-310.
ROWLERSON, A. (1979). Differentiation of muscle fibre types in fetal and young rats studied
with a labelled antibody to slow myosin. /. Physiol. 301,19P.
RUBINSTEIN, N., MABUCHI, K., PEPE, F., SALMONS, S., GERGELY, S. & SRETER, F. (1978). Use
of type specific antimyosins to demonstrate the transformation of individualfibersin chronically stimulated rabbit fast muscles. J. Cell Biol. 79, 252-261.
RUBINSTEIN, N. A. & HOLTZER, H. (1979). Fast and slow muscle in tissue culture synthesize
only fast myosin. Nature 280, 323-325.
SALMONS, S. & VRBOVA, G. (1969). The influence of activity on some contractile characteristics
of mammalian fast and slow muscles. /. Physiol. 201, 535-549.
SALMONS, S. & SRETER, F. A. (1976). Significance of impulse activity in the transformation of
skeletal muscle type. Nature 263, 30-34.
SHAFIQ, S. A., ASIEDU, S. A. & MILHORAT, T. (1972). Effect of neonatal neurectomy on
differentiation of fiber types in rat skeletal muscle. Expl Neurol. 35, 529-540.
STEIN, J. M. & PADYKULA, H. A. (1962). Histochemical classification of individual skeletal
muscle fibers of the rat. Am. J. Anat. 110, 103-124.
THOMPSON, W., KUFFLER, D. P. & JANSEN, J. K. S. (1979). The effect of prolonged, reversible
block of nerve impulses on the elimination of polyneuronal innervation of new-born rat
skeletal muscle fibres. Neuroscience 4, 271-281.
WEEDS, A. G., TRENTHAM, D. R., KEAN, C. J. C. & BULLER, A. J. (1974). Myosin from crossreinnervated cat muscles. Nature 247, 135-139.
WEEDS, A. G., HALL, R. & SPURWAY, N. C. S. (1975). Characterization of myosin light chains
from histochemically identified fibres of rabbit muscles. FEBS Lett. 49, 320-324.
WHALEN, R. G., BUTLER-BROWNE, G. S. & GROS, F. (1978). Identification of a novel form of
myosin light chain present in embryonic muscle tissue and cultured muscle cells. J. molec.
Biol. 126, 415-431.
WHALEN, R. G., SCHWARTZ, K., BOUVERET, P., SELL, S. M. & GROS, F. (1979). Contractile
protein isozymes in muscle development: Identification of an embryonic form of myosin
heavy chain. Proc. natn. Acad. ScL, U.S.A. 76, 5197-5201.
(Accepted 15 March 1983)