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thesis entitled

THE DIFFERENTIATION AND DEVELOPMENT
OF MUSCLE FIBER TYPES IN THE PLANTARIS
OF 1 - 36 DAY OLD RATS

presented by

Jenifer Jo Van Huss Lyster

has been accepted towards fulfillment
of the requirements for

 

 

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THE DIFFERENTIATION AND DEVELOPMENT
OF MUSCLE FIBER TYPES IN THE PLANTARIS
OF 1-36 DAY OLD RATS

By

Jenifer Jo Van Huss Lyster

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Anatomy

1982

ABSTRACT
THE DIFFERENTIATION AND DEVELOPMENT

OF MUSCLE FIBER TYPES IN THE PLANTARIS
OF 1-36 DAY OLD RATS

BY

Jenifer Jo Van Huss Lyster

The purpose of this study was to determine the differ-
entiation and development patterns of the various muscle
fiber types in the plantaris muscle of the rat, from one to
thirty-six days of age. Forty-eight Sprague-Dawley rats
were sacrificed at five-day intervals. The gastrocnemius,
soleus, and plantaris complex was excised and prepared via
fast freezing, for sectioning on a cryostat microtome. The
muscle sections were stained with haematoxylin and eosin to
study morphology. For histochemical analysis, the sections
were stained with myosin adenosine triphosphatase (pH 9.4)
with alkali (pH 10.4) preincubation, and succinate dehydro-
genase. Fiber-to-fiber analysis was performed on the two
histochemical stains, and classified using the nomenclature
of Brooke and Kaiser (1970).

There were three major findings of the study. First is
that muscle fibers are basically undifferentiated at birth.
Secondly, that the differentiation process of the plantaris
muscle follows a smooth and continuous pattern. Last is
that the differentiation process appears to be associated

with the aging process.

Dedication is made to my husband,
Michael, for his love, support, and un-
ending confidence in my ability. And to
my parents, Dr. and Mrs. Wayne D. Van Russ,
and my sisters, Terry, Randie, Trudy, and
Amy, whose patience and love gave me added

purpose and encouragement to complete the

task.

ii

ACKNOWLEDGMENTS

I wish to express my sincere appreciation to my
committee, Dr. R. E. Carrow, chairman, and Dr. R. Echt,
Dr. W. W. Heusner, and Dr. K. W. Ho, for their continued
encouragement, advice, and ceaseless assistance during my
graduate program. I will always be thankful for the

opportunity to study under their trusted guidance.

iii

TABLE OF CONTENTS

INTRODUCTION TO THE PROBLEM.

REVIEW OF THE LITERATURE

METHODS AND MATERIALS.

PRESENTATION OF DATA .

SUMMARY, CONCLUSIONS AND RECOMMENDATIONS .
APPENDICES

A - RAW DATA -- NUMBER OF FIBERS PER FIBER TYPE.

B - PERCENTAGES OF FIBER PER FIBER TYPE.
LITERATURE CITED .

iv

Page

18
22
30

33
35
37

LIST OF TABLES

Table Page

1. Classification of Fiber Types. . . . . . . . . . 21

LIST OF FIGURES
Figure Page

1. Fiber type composition of the plantaris
muscle as a function of age. . . . . . . . . . . 23

vi

INTRODUCTION TO THE PROBLEM

It is well established that skeletal muscle fibers have
been classified into types according to their morphological,
physiological, biochemical and histochemical characteristics.
The fiber types in different mature muscles have been
described for several species (Ariano §£_al., 1973; Peter
gt_a1., 1972). However, the muscle fiber composition profile
of mature muscle is not static (Goldspink and Ward, 1979;
Rowe and Goldspink, 1969; White g£_al., 1978) and can change
throughout life.

It has been shown that cross reinnervation of denervated
muscles can alter the histochemical and biochemical composi-
tion of muscle (Brooke g£_al., 1971). Further evidence is
accumulating that different types of physiological stress on
skeletal muscle alters fiber composition and size (Goldspink
and Ward, 1979; Gonyea g£_al., 1978; Maxwell g£_gl., 1973).
Goldspink and Ward (1979) and Gonyea gt_al, (1978) studied
the response of the different fiber types to high intensity
exercise (i.e., weightlifting). This type of exercise
resulted in hypertrophy of all the fiber types; however,
the extent of the response varied according to the fiber
type. Fibers high in myosin adenosine triphosphatase

activity (type II A, type II B) (Brooke and Kaiser, 1970)

were found to hypertrophy more readily than fibers that were
low in myosin adenosine triphosphatase (type I). Distinction
was made between type II A and type II B, according to the
amount of hypertrOphy, where type II B fibers were found to
hypertrOphy more than type II A. The difference between
these two high myosin ATPase fibers is in their metabolic
enzymes, where type II B is more glycolytic and type II A is
more oxidative.

Maxwell g£_al. (1973) found that with endurance training
the plantaris muscles of guinea pigs at six weeks old, had a
greater proportion of type II A fibers and a lower proportion
of type II B fibers than did the muscles of control animals
at the same age. They also found that there was no difference
between the control and exercised animals in the proportion
of type I fibers, or in the size of the various fiber types.

It has been shown that undernutrition can change the
proportions of the fiber types (Goldspink and Ward, 1979;
Haltia gt_al., 1978). Haltia g£_al. (1978) found that in rats,
undernourished both pre- and postnatally, histochemical
differentiation was retarded in the extensor digitorum longus.
When pregnant rats were undernourished, their growth was
severely retarded and only myotubes were found in the newborn
skeletal muscles. With prOper nutrition postnatally, large
type I and type II B fibers were seen at five days, and the

histochemical maturation proceeded almost normally at later

stages. However, at all stages there was a prOportional
reduction in fiber and body size.

Goldspink and Ward (1979) also found a decrease in size
of the fiber types with partial starvation in guinea pigs
and mice. Of the fiber types, they found that type II B
showed the largest decrease in size. They also determined
that the effects of undernutrition on the fiber types were
completely reversible. Starvation did not have an effect
on the total number of fibers or the numbers of any fiber type.

There are great similarities and differences in the
development and growth of muscles, not only between indi-
vidual muscles, but also between species (Ariano g£_al.,
1973; Colling-Saltin, 1980; Brooke g£_gl,, 1971; Rowe and
Goldspink, 1969). The main difference is in the time course
for development and differentiation. In rats it is believed
that muscles are basically undifferentiated into fiber types
at birth (Curless and Nelson, 1976; Riley, 1977; Shafiq g£_al,,
1972) and that the fiber population found in the adult rat
is determined by the postnatal differentiation process
(Haltia gt_§l., 1978; Curless and Nelson, 1976; Riley, 1977;
Shafiq gt_al., 1972; Goldspink, 1970). Three stages of
development are known to occur prenatally: the immature
myotube, the mature myotube, and the young myofibril. The
mature myofibrils occur postnatally (Platzer, 1978; Colling-
Saltin, 1980). The young rat is born with a high proportion

of mature myotubes to young myofibrils (Haltia et a1., 1978;

Curless and Nelson, 1976; Shafiq gt_al., 1972). However,
this proportion rapidly changes. Curless and Nelson (1976)
found that in the first day of life sixty to eighty percent
of the fibers in the soleus were in the myotube stage. By
the third day only fifteen to thirty percent were in that
stage. Brooke §t_al, (1971) found two types of fibers,
type I and type II, in the soleus of the rat at two days of
age. This investigator states that the type II fiber found
is a type II C, which is a precursor to all other fiber types.
Again, the percentages of the fiber types changed drastically
at the early stages.

It has been shown that neonatal neurectomy will halt
the differentiation process and the fibers will not develop
from the myotube stage (Riley, 1977; Shafiq g£_al., 1972;
Engel and Karpati, 1968). The results of these studies are
discussed in more detail in the literature review. In the
present investigation, only muscles with normal innervation
were studied.

The problem is complex and the reports vary between
investigators, Species and muscles. One possible reason
for the difference in percentages is that many investigators
used random sampling of fields when classifying muscles.
It is the intent of the present investigation to add infor-
mation regarding the differentiation patterns of the entire
plantaris muscle. It is hoped that the extensive nature of

the present study will serve to reduce the confusion of the

fiber type populations in early life, as previously reported
for the plantaris (Maxwell gt_al., 1973).

Many investigators have studied muscle and its dynamic
properties (Close, 1964), but the major questions still exist
regarding the mechanisms re5ponsible for these changes. It
would appear that to initiate study of these changes one must
first attempt to determine how the various fiber types dif-
ferentiate early in life. Therefore, the purpose of this study
was to determine the differentiation and development patterns
of the various fiber types from birth to thirty-six days of
age in the plantaris muscle of the rat using histochemical
techniques. To avoid sampling error, all individual muscle
fibers across the entire muscle section of the plantaris were
classified. The histochemical nomenclature of Brooke and

Kaiser (1970) was used to classify the fibers.

Statement of the Problem

 

The purpose of the problem was to determine the differ-
entiation and deve10pment patterns of the various muscle
fiber types in the plantaris muscle of the rat from one to

thirty-six days of age using histochemical techniques.

Importance of the Study

 

The importance of the study is to add information
regarding the differentiation and deve10pment patterns of
the entire plantaris muscle. Hopefully, this study will reduce
the confusion of previously reported percentages of fiber types
during postnatal growth by eliminating the error of tissue

sampling.

Limitations of the Study

1. The results of this study are applicable only to the
plantaris muscle of the rat under thirty-six days of age.

2. The results of this study are based primarily on fiber-
to-fiber analysis using only two histochemical staining
techniques: myosin adenosine triphosphatase (pH 9.4), with

a preincubation solution of pH 10.4 and succinate dehydro-
genase.

3. The results of this study are based only on histochemical
analysis of muscle fibers, and not biochemical analysis of

muscle fibers.

Definition of Terms

 

Type I fiber. According to the histochemical classifi-

 

cation of Brooke and Kaiser (1970), the reactivity of a type I
fiber using a myosin adenosine triphosphatase (myosin ATPase,
pH 9.4) stain with the preincubation solution at a pH of 10.4
would be inhibited and, therefore, would yield a very light
stain (cream color). Type I fibers contain high amounts of
oxidative enzymes and stain dark with an oxidative enzyme
stain. The succinate dehydrogenase (SDH) stain was used for
the fiber-to-fiber analysis in this study. The type I fiber
is classified as slow-oxidative (3.0.) by Peter gt_§l, (1972).

Type II A fiber. A type II A fiber has an intermediate

 

to high reaction (staining dark brown) when a myosin ATPase
(pH 9.4) stain with a preincubation solution at pH 10.4 is used.
This fiber will have a very strong reaction with the SDH stain

and will stain even darker than a type I fiber. Peter et a1.

(1972) refer to this fiber type as fast-oxidative-glycolytic

(F.O.G.).
Type II B fiber. The type II B fiber demonstrates the

 

strongest reaction to the myosin ATPase (pH 9.4), with a
preincubation solution of pH 10.4, staining a very dark brown
color. On the other hand, this fiber demonstrates a weak
reaction (stains very light) to oxidative enzyme stains.
Peter gt_al. (1972) have classified this fiber type as fast-
glycolytic (F.G.).

Type II C fiber. This fiber stains dark brown with a

 

myosin ATPase (pH 9.4) stain, with a preincubation solution
at a pH of 10.4, and reacts strongly to oxidative enzyme
stains such as SDH. Generally, this fiber cannot be
distinguished using just these two stains and usually is
classified as type II A. Brooke and Kaiser (1970) state
that a type II C fiber is a precursor for the other three
types of fibers. This fiber will not be classified in the
present investigation.

Unidentified fiber. Some fibers cannot be classified via

 

fiber-to-fiber analysis using succinate dehydrogenase (SDH)
and myosin adenosine triphosphatase (myosin ATPase, pH 9.4),
with an alkali preincubation solution. In the present

investigation, these few fibers are listed as unidentified.

REVIEW OF THE LITERATURE

The purpose of this study was to determine the differ-
entiation and development patterns of the various muscle
fiber types in the plantaris muscle of the rat, from one to
thirty-six days of age, using histochemical techniques. For
this review of the literature, three categories of studies
will be examined: histochemical techniques used for classi-
fying muscle fiber types, muscle fiber types in the plantaris
muscle of the adult rat, and differentiation of fiber types

in deve10ping muscles of the rat.

Classification Techniques

 

Early studies on skeletal muscle categorized fiber
types by color (i.e. red or white) and/or speed of contrac-
tion (i.e. fast or slow). More recent investigations, using
various histochemical techniques, have demonstrated that
fiber types cannot be characterized simply on color or speed
of contraction.

In 1955, Padykula and Herman developed a method to
analyze myosin adenosine triphosphatase (myosin ATPase)
activity in skeletal, smooth, and cardiac muscle. This
investigation concentrated on ATPase activity in the myo-

fibrils of skeletal muscle and not the ATPase activity

present in mitochondria. Guth and Samaha (1969) reported
that differences could be determined in the type of ATPase
activity found in skeletal muscle fibers by altering the pH
of the preincubation solution. Myosin ATPase activity is
stable in an alkaline preincubation solution and is labile
in an acid preincubation solution. Guth and Samaha (1969)
found that there were no fibers stable in both acid and
alkaline preincubation solutions.

Brooke and Kaiser (1970) used varying alkali and acid
preincubation solutions to characterize the various types
of fibers for myosin ATPase. They reported two major types,
type I and type II, with type II having three subgroups:
type II A, type II B and type II C. Type I fibers are labile
in an alkali pH and, therefore, demonstrate a weak reaction
when stained for myosin ATPase with a preincubation solution
at pH 10.4. Type II fibers are stable in alkali solutions
and demonstrate a strong reaction to the myosin ATPase stain,
with the preincubation solution at pH 10.4. Type II fibers
show different degrees of lability in various acid solutions
and, therefore, are categorized into three subgroups. The
reaction for type II A fibers is inhibited in a solution of
pH 4.5. For type II B fibers, inhibition is demonstrated at
pH 4.3 and below. Type II C fibers are labile in a solution
of pH 3.9.

The type II C fiber was described further by Brooke
§t_gl. (1971) as the "primitive" fiber. This fiber is

believed to be the precursor fiber to all the other fiber types.

10

Brooke §t_al. (1971) used the same nomenclature to differ-
entiate fibers in the rat from birth to adulthood. Fiber-
to-fiber analysis was performed using the myosin ATPase
stain with alkali and acid preincubation solutions and an
oxidative staining technique. The type II C fiber, when
analyzed histographically for diameter size (Brooke and
Kaiser, 1970) and histochemically via fiber-to-fiber analysis
(Brooke gt_al., 1971), is similar to the type II A fiber.
Therefore, these investigators state that the type II C
fiber usually is included with the type II A fiber when
muscle is analyzed.

Three fiber types, A, B, and C, were reported by Stein
and Padykula (1962) based on the cytochemical distribution
of succinate dehydrogenase (SDH) in the muscle fiber. SDH
is an enzyme which is tightly bound to mitochondria. Fiber-
to-fiber analysis of serial sections of the gastrocnemius
and soleus were performed using SDH, "fixed" and "unfixed"
ATPase, glycogen and other enzyme stains (e.g., non-specific
esterase). These investigators found that the type A fiber
contained few mitochondria and lacked SDH activity in the
subsarcolemma. Type B had more SDH activity and more mito-
chondria than type A. Type C demonstrated the highest
amount of SDH activity and, therefore, the most mitochondria.
This investigation was performed using male and female albino
rats, weighing between 175 and 200 grams. The age of the

animals and the strain of rat were not reported.

11

Peter §t_al, (1972) classified fiber types into three
categories based on the following criteria: the contraction
time of the fiber relative to the other fibers in the same
muscle, the glycolytic capacity of the fiber, and the oxi—
dative capacity of the fiber. The three categories are fast-
glycolytic (FG), fast~oxidative-glycolytic (FOG), and slow-
oxidative (SO). Fast-glycolytic fibers are high in glyco-
lytic enzymes and are fast in contraction. Fast-oxidative-
glycolytic fibers are reported to have the highest concen-
tration of oxidative enzymes with moderate to high amounts
of glycolytic enzymes and to be fast in contraction. Slow-
oxidative fibers have a moderate to high concentration of
oxidative enzymes and are slow in contraction time. These
investigators compared the previously reported nomenclatures
of Brooke and Kaiser (1970), Stein and Padykula (1962) and
others with the nomenclature of PG, FOG, and $0.

The classification of the various types of muscle fibers

has been reported by several authors, each using a different
nomenclature based on different histochemical techniques.
The nomenclature of Stein and Padykula (1962) is not clearly
defined for fiber typing in muscles of rats at an early age.
The nomenclature of Peter gt_al. (1972) involves contraction
time of fibers which is not applicable to this investigation.
Therefore, the nomenclature of Brooke and Kaiser (1970) was

used to classify muscle fiber types in this investigation.

12

Fiber Types in the Plantaris Muscle of the Adult Rat

 

Ariano gt_al. (1973) reported the fiber type populations
for the hindlimb muscles of five mammals. Fiber populations
were estimated histochemically and classified as fast-oxidative-
glycolytic (FOG), fast-glycolytic (PG), and slow-oxidative (80)
(Peter gt_al., 1972). This nomenclature corresponds to that
of Brooke and Kaiser (1970) as type II A, type II B and
type I, respectively. Random sampling of muscle fibers of
serial transverse sections was performed by fiber-to-fiber
analysis of the myosin adenosine triphosphatase and reduced
nicotinamide dinucleotide diaphorase procedures. For the
adult Sprague-Dawley rat, the fiber type profile in the
plantaris muscle was reported as fifty-three percent FOG,
forty-one percent PG, and six percent SO. This profile was
based on a sample size of three rats and a fiber sampling of
381 fibers.

The entire plantaris muscle of six Sprague-Dawley rats,
at 140 days of age, were analyzed histochemically by Ho gt_al.
in 1980. Fiber-to-fiber analysis was performed using the
myosin adenosine triphosphatase (pH 9.4) with the alkali
(pH 10.4) preincubation solution, and the NADH-tetrazolium
reductase stains. These investigators reported a fiber type
composition of forty-seven percent type II A, forty-three
percent type II B and ten percent type I fibers. This fiber
type population agrees reasonably well with the profile
reported by Ariano gt_al. (1973). Total number of fibers in

the plantaris muscle was not reported.

13

The Rat

Many investigators have agreed and reported that muscle
fibers, in the rat, are basically undifferentiated at birth
(Engel and Karpati, 1968; Brooke gt_gl., 1971; Shafiq gt_al.,
1972; Curless and Nelson, 1976; Riley, 1977; Haltia gt_al.,
1978; Ho gt_al., 1980). Reports have varied, however, on
the percentage of fibers in the myotube stage and the ability
to distinguish the fibers at birth. Engel and Karpati (1968)
found that 90 percent of the fibers in the soleus muscle were
in the myotube stage and that the remaining ten percent could
not be differentiated. Shafiq §t_§}, (1972) agreed with these
findings and found this to be true for the gastrocnemius and
extensor digitorum longus muscle also. In both investigations,
the rats were sacrificed within twelve hours after birth.

Haltia gt_al. (1978) report, for one day old animals,
the extensor digitorum longus muscle has 90 percent of the
fibers in the myotube stage, and ten percent are large type I
fibers. Ho gt_al. (1980) report similar results for the
plantaris and soleus muscles at the same age. Haltia gt_al.
(1978) further define the myotubes as being small fetal-type
"F" fibers. They state that the myotubes exhibit similar
staining characteristics to the type II C fibers, which have
been described as the "primitive" fibers by Brooke gt_al. (1971).
These staining characteristics are positive reactions with
both alkali and acid preincubation procedures for the myosin

ATPase (pH 9.4) technique.

14

Brooke gt_al. (1971), using the aforesaid histochemical
techniques, report that myotubes, type I, and type II C fibers
are present at birth in the soleus muscle. Curless and Nelson
(1976) support the finding that fiber differentiation is
evident in one-day-old animals when the myosin ATPase stain
is used with the preincubation solution at pH 4.35. However,
these investigators state that the standard myosin ATPase
procedure fails to reveal any distinct differentiation.

They report that in any one microscopic field 60 to 80 percent
of the fibers may be in the myotube stage. These investiga-
tors report differentiation of the myotube fibers with 90
percent of these fibers being type I and type II C fibers

and the remaining ten percent being of an undetermined fiber
type.

Curless and Nelson (1976) also report that the percen-
tage of type I fibers in the rat soleus remains constant, at
approximately 60 percent (range 53 to 65 percent), from one
to twenty-nine days of age. Even though these investigators
describe the percentage of type I fibers as basically un-
changing with age, they report a dynamic pattern of differ-
entiation for the other fiber types. For example, they
report that the percentage of myotubes per field drops from
60 to 80 percent at one day of age to 15 to 30 percent at
three days of age. The ten percent of undetermined fibers
reported at one day of age are recognized as type II C fibers
at seven days of age. Besides type I fibers, many type II C

fibers are reported to be present until seventeen days of age.

15

Between seventeen and twenty-one days of age, type II C

fibers drop to less than two percent with the type II A
fibers emerging and increasing to thirty-three percent.

At twenty-nine days of age, the fiber profile for the soleus
muscle is reported as 65 percent type I, 33 percent type II A,
and less than two percent type II C.

Several investigators have reported a dramatic decrease,
from birth to between six and ten days of age, in the amount
of myotubes present in muscle regardless of the muscle studied
(Brooke gt_al., 1971; Engel and Karpati, 1968; Shafiq §t_al.,
1972; Curless and Nelson, 1976; Riley, 1977; Haltia gt_al.,
1978; Ho gt_al., 1980). Engel and Karpati (1968) and Haltia
gt_al. (1978) reported a total absence of myotubes at twenty-
one days of age. The latter investigators found a constant
change in fiber composition of the extensor digitorum longus
muscle to 60 days of age.

Brooke gt_al. (1971) and Curless and Nelson (1976) found
that, in the soleus muscle, adult fiber profiles are attained
by four weeks of age. These data are in contrast to the
results of Ho gt_al. (1980) and Ariano gt_al, (1973). Ho
gt_al. (1980) found 75 percent type II A and 25 percent
type I fibers at six and eleven days of age. These percen-
tages changed dramatically between eleven and sixteen days
p of age, and this rapid shift continued until thirty-one days
of age. The fiber distribution then was reported as 25
percent type II A and 75 percent type I. These researchers

also reported that the fiber composition continues to change

16

until 140 days of age when there is sixteen percent type

II A fibers and 84 percent type I fibers. This study is in
disagreement with the previously mentioned study of Curless
and Nelson (1976) in which no significant variation in the
percentage of type I fibers from one to twenty-nine days of
age was observed. One possible reason for this discrepancy
in reported data, is that Ho gt_al, (1980) differentiated
the entire soleus muscle; whereas, Curless and Nelson (1976)
chose random fields for sampling.

Ho et_al, (1980) also described a gradual change in
the percentages of the various fiber types, for the plantaris
muscle, from one to thirty-six days of age. At thirty-six
days of age, there were 80 percent type II A, 14 percent
type II B, and six percent type I fibers. Fiber composition
continued to change until it became 47 percent type II A,

43 percent type II B, and ten percent type I fibers at 140
days of age. This fiber profile is supported by the work of
Ariano gt_al. (1973).

It has been shown that fiber type differentiation is
dependent upon neural stimuli (Engel and Karpati, 1968;
Shafiq gt_al., 1972). Engel and Karpati (1968) observed
that following neonatal neurectomy of the sciatic nerve
type II fibers remained in the myotube stage but that type I
fibers did differentiate histochemically. These authors
described the type I fibers as being less dependent on
neuronal "trophic" influences than type II fibers. Shafiq

et a1. (1972) presented evidence, using both histochemical

17

and electron microscopic techniques, that fiber types are
well differentiated in normal gastrocnemius, extensor
digitorum longus, and soleus muscles two to three weeks after
birth but not in denervated muscles.

Brooke et_al. (1971) demonstrated that with cross re-
innervation of denervated muscles homogenous groups of fibers
are produced. They reported that these uniform groups of
fibers are typical of the innervation of the "foreign" nerve.
Therefore, they concluded that the nerve supply is responsible
for the determination of the fiber type in any given motor unit.

Riley (1977) reported that nearly all muscle fibers of
the soleus are in the myotube stage and are polyneuronally
innervated at birth. He found that the conversion of myo-
tubes to fiber types I and II is dependent on the transition
from polyneuronal to unineuronal innervation. Riley (1977)
also agrees that postnatal denervation halts the differen-

tiation process of muscle fibers.

METHODS AND MATERIALS

Muscles were obtained from forty-eight male albino
rats, Sprague-Dawley*, which were killed at one, six, eleven,
sixteen, twenty-one, twenty-six, thirty-one, and thirty-six
days of age. There were six animals per age group. At
sacrifice, the animals were anesthetized by an intraperitoneal
injection of sodium pentobarbital. The skin was stripped
from foot to thigh and the gastrocnemius, plantaris, and
soleus complex of the right leg was surgically separated
from the rest of the leg. The proximal and distal attachments
of these muscles were held with a hemostat to eliminate
excess twitching. Using scissors, the muscles were detached
and then immediately prepared for freezing.

The muscle complex was attached to a piece of cork,
using gum tragacanth, with the proximal end up. This was
placed in an isopentane solution, precooled to -l70°C with
liquid nitrogen, for quick freezing. The muscle complex
remained in this solution for approximately one to two
minutes. It then was removed from the solution and placed
on a precooled (with liquid nitrogen) aluminum foil square

which was folded for storage. The muscles were stored in a

 

*Spartan Research Animal, Inc., Lansing, Michigan

18

19

freezer at -70°C until they could be cut with a cryostat
microtome and stained histochemically. The same procedure
was followed for the left gastrocnemius, plantaris, and
soleus complex.

Serial transverse sections (8km) were cut with a cryo-
state microtome from the middle of the muscle belly and
placed on coverslips. All sections were rapidly thawed by
placing a warm finger on the underneath side of the coverslips.
Each section was placed in a different coverslip jar that was
labeled with its prospective stain. All sections were air
dried in front of an electric fan for one hour before staining.

Sections stained with haematoxylin and eosin (H 8 E)
were used to study fiber morphology. Fiber type differen-
tiation was examined by histochemical staining for myosin
adenosine triphosphatase (myosin ATPase, pH 9.4) (Padykula
and Herman, 1955) with an alkaline (pH 10.4) preincubation
solution (Guth and Samaha, 1969), and succinate dehydrogenase
(SDH) (Stein and Padykula, 1962). After all staining proce-
dures were performed, the coverslips were mounted on glass
slides using protex for the myosin ATPase and H 8 E stains,
and glycerine jelly for the SDH stain.

The series of histochemically stained slides of the
plantaris muscle were projected with a Prado Universal
Microprojector (Ernest Leitz Gmbh Wetzlar) in a darkroom.

The myosin ATPase (pH 9.4) stain with alkali preincubation

was used first to designate fibers. The stain intensity

20

of each individual muscle fiber on the muscle section was
subjectively evaluated using a rating scale of dark, medium,
and light (Peter §t_al,, 1972). This stain was projected on
white paper, and the fibers staining dark were encircled with
a blue felt-tip pen. The medium staining fibers were noted
with a green felt-tip pen, and the light staining fibers
were designated with a red felt-tip pen. All landmarks
(blood vessels, muscle spindles, etc.) were drawn in with a
number two pencil before proceeding with the fiber-to-fiber
analysis. The entire plantaris muscle was outlined using
this procedure.

The SDH stained slide then was used for fiber-to-fiber
analysis. The landmarks noted from the myosin ATPase (pH 9.4)
and H 8 E stained slides were found and the appropriate fibers
to be used for serial analysis were identified. Staining
intensity again was evaluated as dark, medium, and light
(Peter gt_al., 1972) and designated by making a mark in the
middle of the circled fiber. Dark staining fibers were
designated with a blue mark, medium staining fibers with a
green mark, and light staining fibers with a red mark. The
entire muscle was analyzed using this procedure.

The completed muscle picture was labeled with the
animal number, age, muscle name, date started, date completed,
lens power, and right or left leg determination. The
completed picture of the plantaris muscle ranged from four
feet by six feet to six feet by eight feet in size, depending

upon the age of the animal and the subsequent lens power used.

21

The completed picture then was analyzed and the fiber
categories counted. Each of the individual fibers having
a complete rating scale was typed using the classification
system of Brooke and Kaiser (1970). The following table

illustrates the rating scales and classification.

TABLE 1.
CLASSIFICATION OF FIBER TYPES

 

 

 

 

 

1= ib er Typ e s Mcyfrscilne 1223305: Marks Dc}; 1 o r
Type I tight 232:3”
Type II A fiiiifimté°nifiik 3:5:
Type II B ‘33:: kifih‘

 

Each muscle had a very small percentage of "unknown"
fibers that did not fit the classification of Brooke and
Kaiser (1970). This percentage was trivial and therefore
not recorded.

Mean values of the total numbers and percentages of
the different fiber types were calculated for each muscle

in each age group.

PRESENTATION OF DATA

The purpose of this investigation was to study the
differentiation and development patterns of muscle fiber
types, in the plantaris muscle of rats, at five-day inter-
vals, from one to thirty-six days of age. See Appendix A
for presentation of raw data.

In one-day-old rats, two histochemical fiber papulations
could be identified in the plantaris muscle. The majority
of the fibers were in the myotube stage, but rare type I
fibers were observed using the myosin ATPase (pH 9.4) stain
(Brooke and Kaiser, 1970). Quite often, there was little to
no reaction observed within the myotubes when they were
stained for myosin ATPase activity following alkali preincu-
bation. However, when there was a reaction present in the
myotubes with the myosin ATPase (pH 9.4) stain, the color of
the reaction was slightly darker than the color seen within
the type I fibers. There was no reaction present when
staining for SDH activity at this age. Therefore, exact
percentages of myotubes and type I fibers cannot be presented
due to the difficulty in staining for histochemical reactions
in muscle tissue at one day of age. Consequently, when
referring to Figure 1., the abcissa which represents age is
a broken line, and the first age for reported data is at six
days.

22

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24

At six days of age, 99.2 percent of the fibers in the
plantaris stained positively with both the myosin ATPase
(pH 9.4) and the SDH stains. Less than one percent (0.7%)
of the fibers failed to demonstrate a positive reaction with
the myosin ATPase (pH 9.4) stain and demonstrated a medium
reaction with the SDH stain. Therefore, these fibers were
classified as type II A and type I, respectively (Brooke and
Kaiser, 1970). Single myotubes were found in the periphery
of fascicles. These amounted to 0.1 percent of the fibers
across the entire muscle. The reactions and staining inten-
sities for both the myosin ATPase (pH 9.4) stain and the SDH
stain were not as strong as they were in muscles of animals
at older ages.

The staining intensities of the myosin ATPase (pH 9.4),
and SDH reactions were stronger by eleven days of age, and
there were four fiber populations present (Figure 1.). Only
0.44 percent of the fibers were present in the myotube stage.
Ninety-seven percent were type II A fibers which reacted
strongly with both the myosin ATPase (pH 9.4) and the SDH
stains (Brooke and Kaiser, 1970). This strong reaction
resulted in a medium to dark color shown with the myosin
ATPase (pH 9.4) stain and the darkest color with the SDH
stain (Peter et_al., 1972). Approximately 3.1 percent of
the fibers were found to be type I because they did not
react with the myosin ATPase (pH 9.4) stain and demonstrated

a medium reaction with the SDH stain. The fourth fiber type

25

noted at eleven days of age was the type II B fiber. Due

to an increase in the strength of the histochemical reactions
at this age, it was clearly evident that they were present.
The type II B fiber reacts strongly with the myosin ATPase
(pH 9.4) stain giving the appearance of a dark color, and
there is little to no reaction with the SDH stain. There was
0.11 percent type II B fibers found.

At sixteen days of age, there was a complete absence of
fibers in the myotube stage, and only three fiber types were
found. The fiber population was 94.5 percent type II A, 3.1
percent type I, and 1.1 percent type II B. Unfortunately,
1.4 percent of the fibers at this age could not be identified
via the Brooke and Kaiser (1970) nomenclature and are there-
fore referred to as unidentified fibers.

Between sixteen and twenty-one days of age, the percen-
tage of type II A fibers dropped from 94.5 percent to 87.1
percent. The type I fibers increased slightly from 3.1 percent
to 4.3 percent. There was a sharp increase, from 1.1 percent
to 6.7 percent, in the type II B fibers found. Less than one
percent (0.95%) of the fibers could not be classified at
this age.

It is evident in Figure 1. that no appreciable changes
in percentages were detectable between twenty-one and twenty-
six days of age. Types 1, II A, and II B were 3.4, 88.9, and
6.2 percent, respectively, with 1.6 percent unidentified fibers.

Between twenty-six and thirty-one days of age, there

was a decline in the percentage of type II A fibers from 88.9

26

percent to 82.9 percent. There also was an increase, from
6.2 percent to 10.7 percent, in type II B fibers. There
was a slight decrease, from 3.4 percent to 2.3 percent, in
the percentage of type I fibers. Only 4.2 percent of the
fibers were unidentified at this age.

At thirty-six days of age, 79.5 percent type II A fibers,
14.2 percent type II B fibers and 5.6 percent type I fibers
were found. There were 0.76 percent unidentified fibers.

The histochemical staining reactions for the various
fiber types identified in this investigation are in agreement
with the nomenclature of Brooke and Kaiser (1970). The color
of the staining intensities were subjectively rated using
the system of Peter gt_al. (1972). Table 1. presented in
Methods and Materials was followed for the fiber-to-fiber
analysis of the muscle tissue.

The results of this investigation support the observa-
tion that fiber types are basically undifferentiated at
birth (Riley, 1977; Haltia gt_al., 1978; Shafiq gt_al., 1972;
Engel and Karpati, 1968). Generally, the myotubes failed to
show any selective activity towards the histochemical staining
techniques used. However, in one-day-old animals, a small
proportion of the myotubes demonstrated a slightly darker
reaction with the myosin ATPase (pH 9.4) stain than did the
type I fibers. These results are similar to the staining
reaction found with the type II fibers. However, these fibers

cannot be reported as a type II A or type II B fibers with

27

certainty because they failed to show any reaction with the
SDH technique at this age.

Several investigators have claimed that there is a fiber
present during the early developmental stages in rat muscle
which is a "precursor" to all of the other fiber types. This
fiber is the type II C (or type F) fiber (Curless and Nelson,
1976; Haltia §t_gl,, 1978; Brooke and Kaiser, 1970; Ariano
gt_al., 1973; Brooke gt_al,, 1971). For certain identification
of the type II C fiber one must use the myosin ATPase stain
with acid (pH 4.35) preincubation, but this was not used in
the present investigation. The large fiber population (99.2%)
that demonstrated positive reactions with both the myosin
ATPase (pH 9.4) and SDH stains, at six days of age, may well
stem from this transitional fiber type II C. Although posi-
tive reactions of both stains are highly suggestive of the
type II C fiber, due to not using the myosin ATPase (pH 4.35)
stain for the fiber-to-fiber analysis this population is
reported here as type II A.

The emergence of the type II B fiber was noted at
eleven days of age. The differentiation patterns exhibited
distinct changes starting at sixteen days of age and continued
to thirty-six days of age. The developmental patterns followed
a course which showed a decline in the percentage of type II A
fibers and a steady rise in the percentage of type II B, with
the type I fibers reaching a plateau. Ariano gt_al. (1973)

reports that in the adult Sprague-Dawley rat the fiber type

28

population is fifty—three percent type II A, forty-one
percent type II B, and six percent type I. Ho‘gtygl. (1980)
reports that for rats at 140 days of age the fiber type
population is 47.4 percent type II A, 43.4 percent type II B,
and 9.6 percent type I. Therefore, the differentiation
pattern after thirty-six days of age must be similar to the
pattern established from sixteen to thirty-six days of age

as was found in this investigation. The same general differ-
entiation pattern was observed by Maxwell E£_Elx (1973) in
the plantaris muscle of the guinea pig.

The oxidative enzyme capacity of the plantaris, measured
by the SDH stain, is not apparent and is essentially insig-
nificant at birth. However, oxidative enzyme activity
quickly develops between one and six days of age, and at six
days some SDH activity is demonstrated. The SDH reaction
continues to become stronger with an increase in age. Goldspink
(1969) found that, in muscle tissue of mice, some SDH activity
was noted in larger fibers at birth, but that distinct stain-
ing of the enzyme's activity started at one week of age.
Goldspink also States that differences in staining intensi-
ties are related not only to difference in contents, but also
to changes in growth with age. Maxwell g£_gl. (1973) found
that fibers are relatively undifferentiated on the basis of
SDHFactivity in the plantaris muscle of guinea pigs at one
to two weeks of age. Colling-Saltin (1980) also reports that
there is no oxidative enzyme differentiation in human muscle at

birth and that this development occurs postnatally.

29

Morphologically, many of the myotubes at one and six
days of age appeared to be horse-shoe shaped. At eleven
days old, the myotubes appeared round. Curless and Nelson
(1976) found in the soleus muscle of rats the myotubes
appeared horse-shoe shaped until nine days of age. The large
type I fibers noted at birth, and the type II A and type II
B fibers that appeared later, all demonstrated centrally
located nuclei. By twenty-one days of age, the nuclei
appeared to be located at the periphery of the muscle fiber.
Muscle spindles were first noted at six days of age. The
H G E stain was used for morphological examination of the
muscles. These findings are supported by several investiga-
tors (Curless and Nelson, 1976; Goldspink, 1970; Engel
and Karpati, 1968).

It is evident that although myotubes can be observed
at one day of age, it is not possible to classify fiber types
because they are basically undifferentiated. From six
through thirty-six days of age, there is a progressive
decrease in type II A fibers (from K = 99.2 to K = 79.5 percent)
along with progressive increases in type II B fibers (from
zero to‘K = 14.2 percent) and type I fibers (from X = 0.7
to K = 5.6 percent). Differentiation, however, is only
partially completed by thirty-six days of age as at 140 days
of age the following means are observed: type II A'- 47.4%,
type II B - 43.4%, and type I - 9.6% (Ho gt_gl., 1980). The
differentiation process appears to be associated with the
aging process. The stimuli for producing differentiation

cannot be identified from the present study.

SUMMARY, CONCLUSIONS AND RECOMMENDATIONS

Summary

The purpose of this study was to determine the differ-
entiation and development patterns of the various muscle
fiber types in the plantaris muscle of the rat, from one to
thirty-six days of age, using histochemical techniques.
Previous investigators have reported dissimilar results,
also using histochemical techniques, in either rat muscle or
the plantaris muscle of a different animal. One reason
these reports vary is that many investigators have used
random sampling of the microscopic field when classifying
fiber types. To reduce confusion this investigation classi-
fied the entire plantaris muscle. The intent of this study
was to add information concerning muscle fiber differentia-
tion patterns during the early life of the rat.

Muscles were obtained from forty-eight Sprague-Dawley
rats at one, six, eleven, sixteen, twenty-one, twenty-six,
thirty-one, and thirty-six days of age. There were six rats
per age group. The gastrocnemius, soleus, and plantaris
complex was excised from the leg and prepared via fast
freezing, for sectioning on a cryostat microtome. Serial
transverse sections were cut at 8km in thickness from the

middle of the muscle belly. The sections were stained with

30

31

haematoxylin and eosin (H G E) to study fiber morphology.
Muscle fiber type differentiation was examined by histo-
chemical staining for myosin adenosine triphosphatase (myo-
sin ATPase, pH 9.4) with alkali (pH 10.4) preincubation
solution, and succinate dehydrogenase (SDH).

Fiber-to—fiber analysis was performed on the plantaris
muscle using the myosin ATPase (pH 9.4) stain with alkali
preincubation and the SDH stain. When classifying the
muscle fiber types, the nomenclature of Brooke and Kaiser
(1970) was used with the rating system of Peter gt_al. (1972)
(Table 1.). The entire plantaris muscle was classified at
each age into myotubes, type I, type II A, and type II B
fibers (Figure 1.). The type II C fiber was not classified
in this investigation. Any muscle fibers which did not fit
the nomenclature of Brooke and Kaiser (1970) were classified

as "unidentified" (Appendix A -- Raw Data).

Conclusion

 

Muscle fiber types in the plantaris muscle are basically
undifferentiated at birth, and the differentiation process
occurs postnatally. However, this process is not complete
by thirty-six days of age. The fiber populations continue
to change until adulthood.

The development process seems to follow a smooth pattern
of differentiation. Myotubes and a few large type I fibers
are seen at one day of age. From six to thirty-six days of

age, there is a progressive increase in type I fibers (from

32

X = 0.7 to K'= 5.6 percent) and type II B fibers (from zero
to K = 14.2 percent) with a progressive decrease in type II A
fibers (from K = 99.2 to K'= 79.5 percent). The differentia-

tion process appears to be associated with the aging process.

Recommendations

 

In the future more stains should be performed.

In the future more sections should be cut and stained
following the same histochemical procedures.

In future studies a fiber-to-fiber-to-fiber analysis,
using the myosin ATPase with the acid preincubation solution
should be performed, so as to find type II C fibers.

In future studies all fibers should be counted at birth,

so as to add information about hyperplasia.

APPENDICES

APPENDIX A
RAW DATA -- NUMBER OF FIBERS PER FIBER TYPE

RAW DATA ~- NUMBER OF FIBERS PER FIBER TYPE

 

 

ANIMAL TYPE TYPE TYPE UNIDEN-
NUMBER I II A II B MYOTUBES TIFIED TOTAL
LG-l N/C* N/C 1298**
H-l N/C N/C 1163**
I-l N/C N/C 1818**
K-l N/C N/C 1238**
LK-1 N/C N/C 1104**
L-l N/C N/C 2158**
G-6 95 4558 1 32 0 4686
H-6 26 3661 0 N/C 0 3687
I-6 26 3544 0 N/C 0 3570
J-6 0 3398 0 N/C O 3398
K-6 13 4531 0 N/C 0 4549
L-6 12 4629 0 N/C 0 4641
A-11 13 2881 0 0 0 2894
C-11 507 6289 39 16 0 6851
E-11 102 5419 0 0 0 5521
G-11 62 3715 0 0 0 3777
H-11 106 3892 5 13 0 4016
I-11 182 4621 0 0 0 4203
A-16 193 4344 12 0 1 4550
B-16 216 4584 25 0 2 4827
C-16 105 4683 87 0 129 5004
D-16 124 4335 60 0 102 4621
E-16 168 5323 26 0 35 5552
F-16 101 4733 108 0 136 5078
A-21 155 4179 368 0 36 5038
B-21 165 4034 118 0 95 4412
C-21 149 2568 291 0 4 3012
D-21 278 .5305 244 0 18 5845
E-21 119 2535 156 0 58 2868
F-21 205 3425 438 0 15 4083
B-26 186 4618 259 0 25 5088
C-26 191 3359 256 0 23 3829
D-26 179 3794 401 0 71 4445
E-26 132 3421 206 0 112 3871
F-26 148 3865 224 0 86 4323
G-26 22 2020 124 0 39 2205

*Myotubes and type I present throughout but not counted
except as total.

**Estimated number, counting type I fibers and myotubes.

33

RAW DATA -- NUMBER OF FIBERS PER FIBER TYPE

 

 

ANIMAL TYPE TYPE TYPE UNIDEN-

NUMBER I II A II B MYOTUBES TIFIED ’ TOTAL
A-31 114 3134 401 0 150 3799
B-31 165 3251 381 0 81 3878
C-31 92 3867 259 0 215 4433
D-31 105 3466 382 0 193 4146
E-31 62 4137 780 0 249 5228
F-31 49 4316 724 0 251 5340
A-36 214 3165 238 0 11 3628
B-36 238 3892 547 0 13 4690
C-36 263 4089 763 0 20 5135
D-36 235 3511 814 0 39 4599
E-36 265 3713 895 0 26 4899
F-36 159 1768 377 0 53 2357

APPENDIX B
PERCENTAGES OF FIBERS PER FIBER TYPE

PERCENTAGES OF FIBERS PER FIBER TYPE

ANIMAL TYPE TYPE TYPE

 

NUMBER I II A II B MYOTUBES UNIDENTIFIED

G-6 2.02 97.3 0.0* 0.68 0.0

H-6 0.71 99.3 0.0 0.0 0.0

I-6 0.73 99.3 0.0 0.0 0.0

J-6 0.0 100.0 0.0 0.0 0.0

K-6 0.29 99.7 0.0 0.0 0.0

_ L-6 0.26 99.7 0.0 0.0 0.0
x 6 day 0.67 99.2 0.0 0.1 0.0
A-ll 0.45 99.6 0.0 0.0 0.0
c-11 7.40 91.8 0.57 2.34 0.0
E-ll 1.85 98.2 0.0 0.0 0.0
0-11 1.64 98.4 0.0 0.0 0.0
H-11 2.63 96.9 0.1 0.3 0.0

_ I-ll 4.33 95.7 0.0 0.0 0.0
x 11 day 3.05 96.7 0.1 0.44 0.0
A-l6 4.24 95.5 0.26 0.0 0.0
B-l6 4.47 95.0 0.52 0.0 0.0*
C-16 2.10 93.6 1.74 0.0 2.58
D-16 2.68 93.8 1.30 0.0 2.21
E-16 3.03 95.9 0.47 0.0 0.63
F-l6 1.99 93.2 2.63 0.0 2.13

x 16 day 3.10 94.5 1.07 0.0 1.35
A-21 3.08 83.0 7.30 0.0 0.71
B-21 3.74 91.4 2.67 0.0 2.15
c-21 4.95 85.3 9.66 0.0 0.13
0-21 4.76 90.8 4.47 0.0 0.31
E-Zl 4.15 88.4 5.44 0.0 2.02

_ F-21 5.02 83.9 10.72 0.0 0.37
x 21 day 4.28 87.1 6.66 0.0 0.95
B-26 3.66 90.8 5.09 0.0 0.49
C-26 4.99 87.7 6.69 0.0 0.60
D-26 4.03 85.4 9.02 0.0 1.60
E-26 3.41 88.3 5.32 0.0 2.89
F—26 3.42 89.4 5.18 0.0 1.99

_ G-26 1.00 91.6 5.62 0.0 1.77
x 26 day 3.42 88.9 6.15 0.0 1.56

 

*Less than 0.01 percent.

35

36

PERCENTAGES OF FIBERS PER FIBER TYPE

 

 

ANIMAL TYPE TYPE TYPE
NUMBER I II A II B MYOTUBES UNIDENTIFIED
A-31 3.00 82.5 10.55 0.0 3.95
B-31 4.25 83.8 9.82 0.0 2.09
C-31 2.08 87.2 5.84 0.0 4.85
D-31 2.53 83.6 9.21 0.0 4.66
E-31 1.19 79.1 14.92 0.0 4.76
F-31 0.92 80.8 13.56 0.0 4.70
31 day 2.33 82.9 10.65 0.0 4.17
A-36 5.89 87.2 6.56 0.0 0.30
B-36 5.07 83.0 11.66 0.0 0.28
C-36 5.12 79.6 14.86 0.0 0.39
D-36 5.11 76.3 17.70 0.0 0.85
E-36 5.41 75.8 18.27 0.0 0.53
_ F-36 6.75 75.0 16.00 0.0 2.25
36 day 5.56 79.5 14.17 0.0 0.76

LITERATURE CITED

LITERATURE CITED

Adams, R. 1975. Disease in Muscle: A Study in Pathology.
New York: Harper and Row.

Ariano, M. A., R. B. Armstrong, and V. R. Edgerton. 1973.
Hindlimb Muscle Fiber POpulations of Five Mammals.
J. Histochem. Cytochem. 21:51.

Brooke, M. H., and K. K. Kaiser. 1970. Muscle Fiber Types:
How Many and What Kind? Arch Neurol. 23:369.

Brooke, M. H., E. Williamson, and K. K. Kaiser. 1971. The
Behavior of Four Fiber Types in Developing and
Reinnervated Muscle. Arch Neurol. 25:360.

Brown, M. C., J. K. S. Jansen, and D. Van Essen. 1976.
Polyneuronal Innervation of Skeletal Muscle in

New-born Rats and its Elimination during Maturation.
J. Physiol. 261:387.

Close, R. 1964. Dynamic Properties of Fast and Slow Muscles
of the Rat During Deve10pment. J. Physiol. 173:74.

Close, R. 1967. Properties of Motor Units in Fast and Slow
Skeletal Muscles of the Rat. J. Physiol. 193:45.

Colling-Saltin, A. S. 1980. Skeletal Muscle Development in
the Human Fetus and during Childhood. Children and
Exercise, IX. Baltimore: University Park Press.

Curless, R. G., and M. B. Nelson. 1976. Developmental
patterns of rat muscle histochemistry. J. Embroyol.
exp. Morph. 36, 2:355.

Devlin, R. B., and C. P. Emerson. 1978. Coordinate
Regulation of Contractile Protein Synthesis during
Myoblast Differentiation. Cell. 13:599.

Diamond, J., and R. Miledi. 1962. A Study of Foeta1‘and
New-born Rat Muscle Fibers. J. Physiol. 162:393.

Engel, W. L., and G. Karpati. 1968. Impaired Skeletal

Muscle Maturation following Neonatal Neurectomy.
Dev. Bio. 17:713.

37

38

Goldspink, G. 1969. Succinic Dehydrogenase Content of
Individual Muscle Fibers at Different Ages and
Stages of Growth. Life Sci. 8, 11:791.

Goldspink, G. 1970. The Proliferation of Myofibrils
during Muscle Fiber Growth. J. Cell Sci. 6:593.

Goldspink, G., and P. S. Ward. 1979. Changes in Rodent
Muscle Fibre Types during Post-Natal Growth, Under-
nutrition and Exercise. J. Physiol. 296:453.

Gonyea, W., and F. Bonde-Peterson. 1978. Alterations in
Muscle Contractile Properties and Fiber Composition
after Weightlifting Exercise in Cats. Exp. Neurol.
59:75.

Guth, L., and F. J. Samaha. 1969. Qualitative Differences
Between Actomyosin ATPase of Slow and Fast Mammalian
Muscle. Exp. Neurol. 25:138.

Haltia, M., 0. Berlin, H. Schucht, and P. Sourander. 1978.
Postnatal Differentiation and Growth of Skeletal
Muscle Fibers in Normal and Undernourished Rats. J.
of Neurol. Sci. 36:25.

Hitchcock, S. E. 1970. The Appearance of a Functioning
Contractile Apparatus in Developing Muscle. Dev. Bio.
23:399.

Ho, K. W., W. Heusner, J. Van Huss, and W. Van Huss. 1980.
Postnatal Muscle Fiber Differentiation in Soleus and
Plantaris Muscles of Rats. Med. and Sci. Sports
Exercise. 12, 2:114.

Huizar, P., M. Kuno, and Y. Miyata. 1975. Differentiation
of Motorneurones and Skeletal Muscles in Kittens.
J. Physiol. 252:465.

Korneluissen, H., and J. K. S. Jansen. 1976. Morphological
aspects of the elimination of polyneuronal innervation
of skeletal muscle fibres in newborn rats. J. of

Neurocytology. 5:591.

Mark, R. F. 1980. Synaptic Repression at Neuromuscular
Junctions. Physiol. Reviews. 60, 2:355.

' Maxwell, L. C., J. A. Faulkner, and D. A. Lieberman. 1973.
Histochemical manifestations of age and endurance
training in skeletal muscle fibers. Am. J. of Physiol.
224, 2:356.

39

O'Brien, R. A. D., A. J. C. Ostberg, and G. Vrbova. 1978.
Observations on the Elimination of Polyneuronal
Innervation in Developing Mammalian Skeletal Muscle.
J. Physiol. 282:571.

Padykula, H. A., and E. Herman. 1955. The Specificity of the
Histochemical Method for Adenosine Triphosphatase.
J. Histochem. Cytochem. 3:170.

Peter, J. B., R. J. Barnard, V. R. Edgerton, G. A. Gillespie,
and K. E. Stempel. 1972. Metabolic Profiles of Three
Fiber Types of Skeletal Muscle in Guinea Pigs and
Rabbits. Biochemistry. 11:2627.

Platzer, H. C. 1978. The Ultrastructure of Normal Myogensis
in the Limb of the Mouse. Anat. Rec. 190:639.

Redfern, P. A. 1970. Neuromuscular Transmission in New-born
Rats. J. Physiol. 209:701.

Riley, D. A. 1977. Spontaneous Elimination of Nerve Terminals
from the Endplates of Developing Skeletal Myofibers.
Brain Res. 134:279.

Rowe, R. W. D., and G. Goldspink. 1969. Muscle fibre growth
in five different muscles in both sexes of mice: 1.
Normal Mice. J. Anat. 104, 3:519.

Shafiq, S. A., S. A. Asiedu, and A. T. Milhorat. 1972.
Effect of Neonatal Neurectomy on Differentiation of

Fiber Types in Rat Skeletal Muscle. Exp. Neurol.
35:529.

Stein, J. M. and H. A. Padykula. 1962. Histochemical
Classification of Individual Skeletal Muscle Fibers
of the Rat. Am J. Anat. 110:103.

Strugalska, M. H., and A. Fidzianska. 1976. Development of
Chequerboard Pattern in Human Foetal Skeletal Muscle.
Folia Histochemica et Cytochemica. l4, 4:275.

Swatland, H. J. 1977. Transitional stages in the histochemical
development of muscle fibers during post-natal growth.
Histochem. J. 9:751.

Troyer, H. 1980. Principles and Techniques of Histochemistry.
Boston: Little, Brown and Company.

White, N. A., M. D. McGavin, and J. E. Smith. 1978. Age
Related Changes in Percentage of Fiber Types and Mean
Fiber Diameters of the Ovine Quadriceps Muscles.

Am. J. of Vet. Res. 39, 8:1297.

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