EFFECTS OF LONG TERM PHYSICAL
TRAINING UPON THE HISTOCHEMISTRY
AND MORPHOLOGY OF THE
VENTRAL MOTOR NEURONS

Thesis for the Degree of Ph. D.
MICHIGAN STATE UNIVERSITY
LeRDY B. GERCHMAN
1968

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thesis entitled

Effects of Long Term Physical Training
Upon the Histochemistry and Morphology
of the Ventral Motor Neurons

 

 

presented by
LeRoy B. Gerchman

I
' has been accepted towards fulfillment I
of the requirements for

Ph.D. degree in Anatomy

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ABSTRACT
EFFECTS OF LONG TERM PHYSICAL TRAINING UPON

THE HISTOCHEMISTRY AND MORPHOLOGY OF THE
VENTRAL MOTOR NEURONS

by LeRoy B. Gerchman

The influence exerted by ventral motor neurons upon
red and white skeletal muscle fiber types is becoming more
apparent. For example, cross-innervation and reinnervation
studies demonstrate the capability of nerves to change the
physiologica1_and histochemical profiles of fiber types
within a muscle. Physiological studies have shown that
axons innervating white muscle fibers conduct at a faster
rate than those of red muscle fibers. Henneman 33 El.
(1965) indicate strongly that the dimensions of motor
neurons are directly related to their susceptibility to
discharge, therefore to theis activity and possibly to the
red and white motor units within a skeletal muscle. It
was, therefore, believed to be of interest to examine the
histochemical and morphological changes which occur in the
ventral motor neuron with a long term exercise program
deSigned to produce changes in the proportions of red to

white muscle fibers in skeletal muscle.

LeRoy B. Gerchman

A number of investigations demonstrate that the
morphology of motor neurons changes with increased
functional activity. Few studies, however, deal with the
influence of physical activity upon the histochemistry of
the motor neuron. In both morphological and histochemical
studies there is often a lack of distinction between the
effects of a sudden exhaustive increase in activity and
the effects of continued muscular training.

The purpose of this study is to determine changes
in morphology as well as to estimate alteration in enzyme
activity in the ventral motor neuron following long term
physical exercise programs.

Sixty, male Sprague-Dawley rats, 100 days of age
were randomly placed into three groups. The first group
was housed in sedentary cages and comprised the sedentary
control group. The second group was housed in sedentary
cages but was subjected daily to a period of forced exer-
cise. The third group was housed in spontaneous exercise
cages and was subjected daily to two periods of forced
exercise. A period of forced exercise consisted of thirty
minutes of swimming with weights attached to the animals'
tails. Each weight equaled three per cent of the animal's
body weight in the second group and four per cent of the
animal's body weight in the third group. The experimental

procedures were carried out for a period of fifty-two days.

LeRoy B. Gerchman

At the time of sacrifice the lumbar intumescence of
the Spinal cord was immediately removed. Fresh frozen
sections were taken for cholinesterase, acid phosphatase,
malate, and glucose-6-phosphate dehydrogenase techniques.
Formalin-fixed, paraffin-embedded sections were taken for
staining with toluidine blue and luxol fast blue-neutral
red.

Areas of cell bodies, nuclei, and nucleoli of
ventral motor neurons were obtained from projected images
of ventral horns with the aid of a compensating planimeter.
Histochemically the motor neurons were graded according to
intensity and the percentages of dark, medium and light
staining cells were recorded for the three groups.

Approximately 2000 ventral motor neurons were
evaluated for each enzyme and for area measurements with-
out knowledge of the experimental groups from which they
came.

Cholinesterase, glucose-6-phosphate dehydrogenase,
and acid phOSphatase showed marked activity changes with
increased functional activity. Alterations in morphology
and staining properties of ventral motor neurons were also
observed at both experimental levels of increased functional

activity.

EFFECTS OF LONG TERM PHYSICAL TRAINING UPON
THE HISTOCHEMISTRY AND MORPHOLOGY OF THE

VENTRAL MOTOR NEURONS

BY

LeRoy B. Gerchman

A THESIS

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

DOCTOR OF PHILOSOPHY
Department of Anatomy

1968

DEDICATED TO:
MY WIFE, VINNY:

For her love, understanding, and
encouragement.

OUR PARENTS:

The most wonderful peOple in the world,
who have contributed so much in so many ways.

OUR CHILDREN:

Who make it all worthwhile.

ii

ACKNOWLEDGMENTS

I am deeply indebted and grateful to my advisor,
Dr. Rexford E. Carrow, for his guidance, help and encour-
agement throughout this study and for the use of
laboratory equipment and facilities.

A special word of thanks is extended to Dr. W. W.
Heusner and Dr. W. VanHuss, Department of Health, Physical
Education and Recreation, for providing animal quarters,
feed, cages and all the exercise equipment required in
this study.

Special thanks are due to Robert Echt for the
acquisition of exPerimental animals utilized in this study
and to Reggie Edgerton for his assistance with statistical
treatment of data. Many thanks are also extended to these
gentlemen for providing an intellectually stimulating
atmosphere in which to work.

A word of appreciation to Dr. R. A. Fennell of the
Department of Zoology for stimulating an interest in histo-
chemistry and for his advice and help during the course of
this study.

A word of appreciation is in order to Dr. Bruce
Walker and Dr. M. Lois Calhoun for the use of departmental

facilities.

iii

Many thanks to Barbara Wheaton for her efficiency
and continual help, as well as for providing a pleasant
laboratory environment.

I would especially like to express my deepest
appreciation and thanks to my wife, Vinny, for her sacri-
fices; and for the many hours spent in typing and retyping

this manuscript.

iv

VITA

LEROY B. GERCHMAN

Candidate for the degree of Doctor of PhilosoPhy

 

Final examination: July 29, 1968. 1:00 p.m.

Dissertation: Effects of long term physical exercise upon

 

the histochemistry and morphology of the
ventral motor neurons.

Major Subject: Anatomy

 

Biographical items:

 

Born: April 28, 1941. Forest City, Pennsylvania.
Undergraduate studies:
8.5., Biology; University of Scranton, Scranton,
Pennsylvania, 1963.
M.S., Anatomy; Creighton University, Omaha,
Nebraska, 1965.

Professional experience:

 

Graduate assistant, Department of Anatomy, College
of Human Medicine, Creighton University, 1962-63.

Graduate assistant, Department of Anatomy, College
of Veterinary Medicine, Michigan State University,

1965-66.

Graduate assistant, Department of Anatomy, College

of Human Medicine, Michigan State University,

1966-68.

Member of Society of Phi Kappa Phi.

vi

TABLE OF CONTENTS

INTRODUCTION 0 O I O O O O O O O O O O O O O O C

General Comments . . . . . . . . . . . .
Development of Problem . . . . . . . . .
Specific Aims of Research . . . . . . .
Significance of Study . . . . . . . . .

REVIEW OF PERTINENT LITERATURE . . . . . . . . .

Red and White Skeletal Muscle . . . . .
Anatomical Differences . . . . . . .
Physiological Differences . . . . .
Biochemical Differences . . . . .

Neural Influence Upon Red and White

Muscle . . . . . . . . . . . . . . . .

The Motor Unit . . . . . . . . . . . . .

Motor Neurons and Fiber Types .-. . . .

Influence of Exercise . . . . . . . . .

Functional Activity and the Motor Neuron
Morphological Effects . . . . . . .
Histochemical Effects . . . . . . .

MATERIAI‘S AND METHODS O O O O O O I O O O O O 0

Experimental Animals . . . . . . . . . .
Experimental Groups . . . . . . . . . .
Experimental Treatment . . . . . . . . .
Tail Weights . . . . . . . . . . . .
Swimming . . . . . . . . . . . . .
Environment and Food . . . . . . . .
Sacrifice Procedures . . . . . . . . . .
Histochemical Procedures . . . . . . . .
Freezing . . . . . . . . . . . . . .
Cutting . . . . . . . . . . . . . .
Incubation . . . . . . . . . . . . .
Histochemical Techniques . . . . . . . .
Cholinesterase . . . . . . . . . . .
Acid Phosphatase . . . . . . . . . .
Malic Acid Dehydrogenase. . .
Glucose- 6- -Phosphate Dehydrogenase .

vii

Page

m sswtuka +4

CDQO‘OW

15
19
21
21
26

31

31
31
32
32
33
34
35
36
36
36
37
37
37
38
39
39

Page

Histological techniques . . . . . . . . . . . 40
Measurements of Neurons . . . . . . . . . . . 40
Evaluation and Analysis . . . . . . . . . . . 41
Histochemical . . . . . . . . . . . . . . 41
Reliability Data . . . . . . . . . . . . 42
Measurement Data . . . . . . . . . . . . 43

RESULTS 0 O O I O O O O O O O O O O O O O O O O O O O 44

Histochemical . . . . . . . . . . . . . . . . 44
Cholinesterase . . . . . . . . . . . . . 44
Glucose-G-Phosphate Dehydrogenase . . . . 48
Acid Phosphate Dehydrogenase . . . . . . 51
Malic Acid Dehydrogenase . . . . . . . . 54
Reliability Data . . . . . . . . . . . . 55

Morphological . . . . . . . . . . . . . . . . 57
Nissl Staining . . . . . . . . . .'. . . 57
Measurements of Neurons . . . . . . . . . 58
Body Weights . . . . . . . . . . . . . . 59

DISCUSSION 0 O O O O O O O O O O O O O O O O O O O O 61

Sedentary-Forced Group . . . . . . . . . . . 62
Voluntary—Forced Group . . . . . . . . . . . 64
Relationship to Muscular Results . . . . . . 67
Limitations of This Study and Suggestions

for Future Studies . . . . . . . . . . . . 71

SUMMARY AND CONCLUSIONS 0 O O O I O O O O O O O O O O 7 3

smary O O O O O O O O O O O O. O O O O O O O 73
Conclusions . . . . . . . . . . . . . . . . . 75

LITERATURE CITED 0 O O O O O O O O O O O O O O O O O 77

APPENDIX 0 O O O O O 0 O O O 0 O O O O 0 O O O O O O 99

viii

TABLE

10.

11.

LIST OF TABLES

Cholinesterase: Mean percentages of dark,
medium, and light staining neurons in
three experimental groups . . . . . . . . .

Frequency distribution of subjective
ratings for Cholinesterase activity in
treatment groups . . . . . . . . . . . . .

Glucose-6-ph05phate dehydrogenase: Mean
percentages of dark, medium, and light
staining neurons in three experimental
groups . . . . . . . . . . . . . . . . . .

Frequency distribution of subjective
ratings for G-6—PD activity in treatment
groups C O O I O O I O O O O O O O O O O 0

Acid phosphatase: Mean percentages of
dark and light staining cells in three

eXperimental groups . . . . . . . . . . . .

Frequency distribution of subjective
ratings for A.Pase activity in treatment
groups I I O O O O O O O O O O O O O O O O

Malic acid dehydrogenase: Mean percentages
of dark, medium, and light staining cells
in three experimental groups . . . . . . .

Frequency distribution of subjective
ratings for MDH activity in treatment
groups 0 O O O O O O O O O O O O O O O O 0

Correlation from reliability data . . . . .

Mean areas of ventral motor neurons in
square micra . . . . . . . . . . . . . . .

Body weights and per cent correlation

between-body weights and mean neuronal
areas 0 O I O O O O O O O O I O O O O O O 0

ix

Page

46

47

49

50

52

53

55

55
57

58

60

10.

ll.

12.

LIST OF FIGURES

Cholinesterase activity in ventral motor
neurons of a sedentary animal . . . . . .

Cholinesterase activity predominant in
the neurons of the sedentary-forced group
Of animals I I I I I I I I I I I I I I I

Ventral motor neurons of the voluntary-
forced group of animals incubated for
Cholinesterase . . . . . . . . . . . . .

G-6-PD activity in ventral motor neurons
of a sedentary animal . . . . . . . . . .

Activity of G—6-PD in motor neurons of
the sedentary-forced group showing in-
creased numbers of light staining cells .

G-6-PD activity in the ventral horn of a
voluntary—forced animal . . . . . . . . .

Acid phOSphatase activity in two neurons
of the sedentary group of animals . . . .

Increased acid phosphatase activity in
neurons of the sedentary-forced group . .

Diminished acid phosphatase activity in
motor neurons of the voluntary-forced
group Of animals ‘ o o o o o o o o o o o o

Nissl staining in ventral motor neurons
of a sedentary animal . . . . . . . . . .

ChromOphobic ventral motor neurons in
the sedentary-forced group . . . . . . .

Severe chromOphilic reactions in neurons
of the voluntary-forced group of animals

Page

90

90

90

92

92

92

94

94

96

96

96

Figures Page

13. CHE activity in a medium staining cell

from a sedentary-forced animal . . . . . . . 98
14. CHE activity in a ventral motor neuron

of a voluntary-forced animal . . . . . . . . 98
15. MDH activity in two motor neurons from

the sedentary group of animals . . . . . . . 98
16. Acid phosphatase in a neuron from the

sedentary group . . . . . . . . . . . . . . 98
17. Toluidine blue stained neuron from the

sedentary-forced group . . . . . . . . . . . 98
18. Toluidine blue stained neuron from the

voluntary—forced group . . . . . . . . . . . 98
19. BuChE in the ventral horn from a

voluntary—forced animal . . . . . . . . . . 98

xi

LI ST OF APPENDICES

Appendix Page
A. Histochemical Evaluation Data .y. . . . . . . 99
B. One Way Analysis of Variance Tables . . . . . 110
C. Neuron Areas and Body Weights . . . . . . . . 116

xii

AChE

A.Pase

BuChE
CHE

DPN

E-600

G-6-PD

MDH

NBT

TPN

LIST OF

ABBREVIATIONS

xiii

Acetycholinesterase
Acid phosphatase
Pseudocholinesterase
Cholinesterase

DiphOSphOpyridine
nucleotide

Di-ethyl-P-nitro-
phenyl phOSphate

Glucose-G-phOSphate
dehydrogenase

Malic acid dehydro-
genase

Nitro blue tetrozolium
salt

Triphosphopyridine
nucleotide

INTRODUCTION

General Comments

 

The present investigation represents the neural
aspects of a complementary neuromuscular study concerned
with the influence of physical exercise programs upon
neuromuscular histochemistry and morphology. The intimate
relationship between nerve and muscle lends itself readily
to a coinvestigation of this type in which the same exper-
imental animals can be utilized and the results of each
study interpreted in a framework of the motor unit rather
than individually. A complete treatment of the muscular
aSpects of this problem is presented in a doctoral thesis
submitted to the Department of Health, Physical Education,

and Recreation (Edgerton, 1968).

Development of Problem

 

That the intact motor neuron exerts a "trOphic"
influence upon the muscle it supplies was probably first
observed clinically following disturbances of innervation.
Denervation studies have now clearly demonstrated a strong
dependance of muscle upon an intact nerve supply (Nachmias
gglal., 1958; Sheves gplal., 1956; Romanul gt 31., 1965;

Humoller et a1., 1951; Hogan gt_al., 1964). More recent

evidence obtained from cross-innervation and reinnervation
studies (Close, 1965; Buller gt 31., 1960b; Romanul 35 31.,
1966; Yellin, 1967) suggest not only a general dependency
between nerve and muscle but a very specific relationship
between a neuron and the muscle fiber type which it innerv-
ates.

Mammalian skeletal muscle can be differentiated into
red and white types based upon anatomical, physiological
and biochemical prOperties. The red and white fibers re-
present the extremes of a continuum with a number of inter-
mediate types as demonstrated by histochemical techniques
(Romanul, 1964). The close relationship which exists
between a neuron and the specific muscle fiber types
it innervates suggests that neurons supplying opposite
types would exhibit distinguishing characteristics. Physio-
logical evidence has revealed the existence of different
kinds of alpha motor neurons based upon their speed of
conduction, the size of their spike potentials, and their
susceptibility to discharge (Granit gt 21., 1956). From
physiological evidence it has been postulated that neurons
innervating red muscle fibers are smaller in size and more
active, in terms of discharge and conduction, than those
supplying white fibers (Somjen et 31., 1964; Henneman 22 a1.,
1965).

There is a considerable amount of physiological

and histochemical evidence which points to a fine control

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of the biochemical and kinetic properties of muscle fiber
types. It was therefore the author's contention that non—
pathological alterations in the prOportions of red to white
fibers in skeletal muscle would be reflected in the enzyme
activity and morphological features of the neurons supply-
ing those muscles.

Heretofore, it has not been demonstrated that an
exercise program would provide a non—pathological means of
altering the prOportions of red to white fiber types in
skeletal muscle. However, the results of certain quantitat-
ive biochemical studies (Halloszy, 1967; Peter gt 21., 1968)
suggest that a possible shift in the proportions of fiber
types rather than an overall change in muscle metabolism
may occur with exercise. Other authors have suggested
that the transformation of fiber types probably occurs as
the result of changing functional demands upon nerve and
muscle (Guth £3 31., 1968). It was therefore believed that
alterations in muscle fiber types would occur, with an ex-
ercise program, and that such changes would be reflected
in the motor neurons of the spinal cord, therefore, the

entire motor unit.

Specific Aims of Research

In view of the influence exerted by motor neurons
upon red and white fiber types it was believed to be of

interest to examine the ventral motor neuron following long

term physical exercise programs designed to produce
alterations in the prOportion of red to white fiber types
in skeletal muscles supplied by those neurons.

The specific aims of this research were first to
investigate the influence of long term physical exercise
programs upon the ventral motor neuron. Histological and
histochemical techniques were utilized in examining the
following parameters (1) the sizes of cell bodies, nuclei
and nucleoli (2) the staining of Nissl substance within
motor neurons (3) the enzyme activity of motor neurons as
demonstrated by histochemical techniques. Secondly, the
findings were to be correlated with the changes in muscle
fiber types obtained by Edgerton (1968) from skeletal

muscle of the same eXperimental animals.

Significance of Study

 

In view of the above discussion it is believed
that this study will contribute to the existing body of
knowledge concerned with the effects of increased func-
tional activity upon motor neurons. In regard to the
convincing evidence on the delicate neural control of the
kinetic and metabolic prOperties of muscle fiber types
this study should further emphasize the intimate relation-
ships between ventral motor neurons and fiber types within
skeletal muscle. It should also contribute much needed

evidence regarding the adaptability of the motor neuron,

as an integral part of the motor unit, to meet the demands

of increased functional activity.

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REVIEW OF PERTINENT LITERATURE

Red and White Skeletal Muscle

Mammalian skeletal muscle fibers can be
differentiated into red and white fiber types. Differences
between the two fiber types can be observed anatomically,
physiologically, and biochemically. The following para-
graphs are intended to highlight the major differences on

all four levels of investigation.

Anatomical

 

That skeletal muscles of animals varied in color
from a deep red to a pale pink color has been known as
early as 1678 (Denny-Brown, 1929a). Early morphological
studies revealed that two major categories of fibers exist
in varying prOportions in the skeletal muscles of mammals.
One fiber type had a smaller diameter and a more granular
sarc0plasm and was described as a dark or red fiber. The
other, a light or white fiber, was larger in diameter with
a less granular sarc0plasm (Bullard, 1912; Denny-Brown,
1929a). Increased quantities of myoglobin and cytochromes
have been shown to be responsible for the red color of the
smaller fibers (Hawrowitz and Harden, 1954). Most mammal-

ian muscles are "mixed" in that they contain both red and

 

I

3
4

hate fibers

 

therefore, do
thmm
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HBO. Rece
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OXidative

section .

red and
muscles
attempt
aCtivity

of red a:

white fibers (Needham, 1926). The term "red" or "white,"
therefore, does not necessarily imply complete homogeneity
of the component fibers.

Increased capillary to fiber ratios have been
demonstrated in red muscle fibers and have been related
to the increased oxidative metabolism of red fibers (Romanul,
1965). Recently, an increase in the capillary to fiber ratio
of both red and white fibers has been demonstrated in exer-
cised rats (Carrow 33 a1., 1967).

Electron microsc0pic studies have revealed a greater
number of mitochondria in red than in white fibers with a
predominantly peripheral distribution of mitochondria in
the red fibers (Gauthier and Padykula, 1966; Gauthier,
1968).

The increased vascularity and increased numbers of
mitochondria in red fibers is consistent with the increased
oxidative metabolism of these fibers discussed in a later

section.

Physiological

 

Early investigators described the contractions of
red and white skeletal muscles and demonstrated that red
muscles contracted more slowly than did the white. In an
attempt to relate histologic structure with functional
activity, Denny-Brown (1929a) described the contractions

of red and white muscles and considered the quota of red

and white fibers in relation to the physiological prOperties
of the whole muscle. Close (1964, 1965, 1967) studied the
contraction characteristics of skeletal muscle and reported
that red fibers contract at a slower rate than white fibers.
From the same work he concluded that slower red fibers are
associated with the sustained contractions generally
associated with postural muscles while white fibers are

responsible for rapid forceful contractions.

Biochemical

 

Data from both histochemical and quantitative
biochemical sources show marked differences in the various
metabolic pathways of red and white muscle fibers. Red
muscle fibers have intense activity of enzymes related to
oxidative and lipid metabolism and represent a strong
dependence upon aerobic pathways. White muscle fibers
demonstrate higher activities of enzymes concerned with
anaerobic glycolysis (Dubowitz e; 21., 1960; Dawson 32 a1.,
1964; Romanul, 1964; Beatty 35.31., 1966). Beatty 31 a1.
(1963) have also shown intense concentrations of glycogen
and phosphorylase in white fibers which is consistent with
a system dependent upon glycolytic metabolism and an
:hmnediately available energy reserve. The basic blochemical
differences between red and white fibers allowed enzyme
histochemistry to become a very valuable tool in the

<iifferentiation of fiber types.. Until recently the main

histochemical methods for differentiating fiber types were
those of Sudan III and IV (Bullard, 1912; Denny-Brown,
1929a). With the descriptions of differences in succinic
dehydrogenase found in the different fiber types (Padykula,
1952; Thimann and Padykula, 1955) the methods of differen-
tiating fiber types shifted toward enzyme histochemistry.
Since these early studies a number of enzyme techniques
have been employed to differentiate muscle fiber types

(Stein and Padykula, 1962; Romanul, 1964).

Neural Influence Upon Red and White Muscle

 

A number of investigations have also demonstrated
the strong dependence of muscle upon an intact nerve supply
to maintain normal contraction characteristics as well as
normal histochemical and biochemical differentiation
(Humoller 33 31., 1951; Humoller 33_31., 1952; Sheves 33
31., 1956; Nachmias 33 31., 1958; Hogan 33 31., 1964;
Close, 1965; Romanul 33 31., 1965; Guth 33 31., 1967). It
has been speculated that perhaps differentiation of muscle
fibers into red and white types, as defined by metabolic
criteria is under the control of motor neurons since both
innervation and metabolic differentiation occur early in
fetal development (Beatty 33 31., 1967).

Utilizing the technique of surgical cross—union

of nerves Buller, Eccles, and Eccles, (1960b) and Close

(1965) demonstrated that a slow contracting muscle,

   
    

mmnervated "
sacked locrea.
the telhherva
muscle reduce
permeate m

that these Ch

 

Mon and Iege
acquired lnne
1nIlervate mus
From these 5
POStUlated T.
prOdUCed tI'lI
motor “euro'
muscles res
Sll
paVe ShOWn
1st‘1c5 hax
contractl:
IDUbowitz
Romanul,
crOSS‘Un:
Changes ‘
PhySiO 10‘
(1966)
Chemical

Gross‘un

10

reinnervated by the motor nerve to a fast muscle, shows a
marked increase in its speed of contraction. Conversely,
the reinnervation of a fast muscle by a nerve to a slow
muscle reduces the speed of its contraction. Control ex-
periments with self union and regeneration demonstrated
that these changes were not merely a result of nerve sec-
tion and regeneration but were the result of a newly
acquired innervation by a motor nerve that normally would
innervate muscle having a different Speed of contraction.
From these studies Buller, Eccles and Eccles (1960b)
postulated that the changed speeds of contractions were
produced through the influences of two types of alpha
motor neurons which normally innervated fast and slow
muscles respectively.

Since studies of enzymes in normal skeletal muscle
have shown that muscles with fast contraction character-
istics have a preponderance of white fiber types and slow
contracting muscles have a preponderance of red fiber types
(Dubowitz and Pearse, 1960; Romanul, 1964; Dawson and
Romanul, 1964), the question arose as to whether surgical
cross-union of motor nerves would produce corresponding
changes in the histochemical profiles of fibers within the
physiologically altered muscles. Romanul and Van Der Meulen
(1966) investigated the physiological as well as the histo-
chemical alterations produced in muscle as a result of

cross-union of motor nerves. The physiological alterations

were in agree
Close (1965)
chemical cha:
capability o:
and histoche:
therefore, bl
union studle
The .
are able to .
biochemical 1
One PrOposal
of impulses l
as the Contr.
between red

ECCles' and
R" 1965) .
not Observed

cats (Bulls:

11

were in agreement with those of Buller 33 31. (1960b) and
Close (1965) and were accompanied by corresponding histo-
chemical changes as confirmed by Yellin (1967a). The
capability of the motor neuron to alter the physiological
and histochemical profiles of red and white muscle has,
therefore, been clearly demonstrated by motor nerve cross-
union studies.

The exact mechanism by which ventral motor neurons
are able to affect alterations in the physiological and
biochemical prOperties of muscle fiber types is unknown.
One prOposal has suggested the sequence or the quantity
of impulses per unit time delivered to the muscle fiber
as the controlling factor. Cross-union of motor nerves
between red and white muscle can reverse the contraction
characteristics of the newly innervated muscles (Buller,
Eccles, and Eccles, 1960b; Buller and Lewis, 1965; Close,
R., 1965). Reversal of contraction characteristics was
not observed when cross-union was performed in spinalized
cats (Buller, Eccles and Eccles, 1960a). Conversely,
continuous stimulation of the pOpliteal nerve consistently
resulted in a slowing of contractions in the anterior
tibialis muscle (Salmons and Vrbova, 1967). Together
these studies indicate the importance of an impulse con—
duction mechanism in altering muscle fiber types.

A second mechanism by which motor neurons are

believed to influence muscle fibers is through the release

 

12

of some chemical factor from the neuron into the muscle
fiber, the "ooze" theory. However no such chemical agent
has yet been identified. There are certain observations,
however, which can readily be eXplained by such a mechan-
ism. For example, at birth all muscles contract slowly;
only shortly after birth do the contraction times of white
muscle shorten (Denny-Brown, 1929a; Buller, Eccles and
Eccles, 1960a; Buller and Lew1s, 1965). This shortening
of contraction times can be reversed by transection of the
peripheral motor nerve but not by transection of the spinal
cord and deafferentation, which silences the ventral motor
neurons. Therefore, the differentiating process appears
to be related to a neuronal control but not to nerve im-
pulses (Buller, Eccles, and Eccles, 1960a).

Activation of glycogen-resynthesis in muscle occurs
after direct stimulation of its motor nerve (Gutmann 33 31.,
1954). After stimulation, sectioning of the nerve produces
an impairment of glycogen-resynthesis in the muscle which
takes place at a time related to the length of the peri-
pheral stump (Gutmann 33 31., 1956). Substances from the
motor neuron released into muscle fibers apparently take
part in the activation of this process. That substances
move proximodistally in the axons of motor nerves has been
well established (Sawyer, 1946; Weiss 33 31., 1948; Hebb
33 a1., 1956; Friede, 1959; Weiss, 1967a; Weiss, 1967b).

Nerve stimulation or physiological activity induces the

uahsport of
substances CO
proximal and
been demonst'
istherefore
agent is lit
mfluences I
shtheSis.
It
sdely in 1
they all h
substance.
IEpresehts
probably I
in skelet
that the

lSthS of

ential e

regard t.

3::

re all

neuron,

they rep

13

transport of substances within axons (Vodicka, 1956). That
substances continue to be transported for a time in the
proximal and distal portions of a severed nerve has also
been demonstrated (Hebb 33 31., 1956; Friede, 1959). It

is therefore apparent from these studies that a neural
agent is liberated from the neuron into the muscle which
influences at least the metabolic process of glycogen
synthesis.

It is not possible to eXplain all observations
solely in terms of electrical activity of nerves, nor can
they all be accounted for by some hypothetical neurotrOphic
substance. Furthermore, it is probable that neither
represents the complete mechanism and that the two are
probably closely interrelated in producing an influence
in skeletal muscle. In any case the important concept is
that the neuron is capable of regulating many character-

istics of muscle fiber types.

The Motor Unit

 

The previous discussion indicates that the prefer-
ential energy metabolism of fiber types is determined by
nerve SUpply. A basic concept which must be discussed in
regard to this idea is the homogeneity of the motor unit.
Are all the muscle fibers, supplied by a specific motor
neuron, of the same histochemical type (homogeneous) or do

they represent both red and white histochemical types

(heterogene
isgovernec
reasonable
heron won
RC
[1967a, b‘
strongly
exception
distribut
ably. H.
to be ra
ing que
or islar
found.
finding
direct
fibers
collate
fIbers
and the
not bee
tOrtuoi
mu5cle
arIIVe
beED in

reSuit

14

(heterogeneous)? If the energy metabolism of fiber types
is governed by the neurons which supply them it would seem
reasonable that all the fibers governed by a specific
neuron would be of the same histochemical type.

Romanul and Van Der Meulen (1966) and Yellin
(1967a, b) have presented histochemical evidence which
strongly suggest homogeneity of the motor unit. With few
exceptions the muscle fibers of a motor unit are widely
distributed in a muscle and motor units overlap consider-
ably. Histochemically red and white fiber types are seen
to be randomly interSpersed between one another. Follow-
ing regeneration of a motor nerve to a muscle, type groups
or islands of enzymatically similar muscle fibers are
found. Romanul and Van Der Meulen (1966) interpret this
finding to mean that during normal development axons follow
direct pathways within a nerve and arrive at the muscle
fibers in close time relationship with other axons. As
collaterals form they are unable to innervate near by
fibers which have already been innervated by adjacent axons
and therefore grow out until they reach fibers which have
not been innervated. Regenerating axons, because of the
tortuous course through the area of trauma, arrive at
muscle fibers at widely varying times. The first axon to
arrive can innervate adjacent fibers which have not yet
been innervated.- Subsequent axons act similarly. The

result being that an axon supplies the muscle fibers in a

soecriic area

histochemica‘

;

unit. it wa
type groups
iibers are

pig Iherkel
would seem
tures in t

muscle.

E'
that red

dEpendem:
and PhYSl
alrlses as
SPECific

units of

in a Sun;
reS‘Pondin
demonsue
at the be
Charged ti
alpha mot<
by their 3

Skoghlnd g

15

specific area and all the fibers supplied by that axon are
histochemically Sim11ar suggesting a homogeneous motor
unit. It was of interest to this author to learn that
type groups or islands of enzymatically similar muscle
fibers are a normal feature in the skeletal muscles of the
pig (Merkel, 1968). In view of the above discussion it
would seem that this animal may exhibit distinctive fea-
tures in the developmental innervation of its skeletal

muscle.

Motor Neurons and Fiber Types

 

Evidence presented in previous discussions suggests
that red and white motor units are, to a large extent,
dependent upon their reSpective neurons for biochemical
and physrological differentiation. The question then
arises as to what characteristics can be attributed to the
SpeC1f1C neurons supplying red, white or intermediate motor
units of skeletal muscle. Granit, Henatsch, and Steg (1956),
in a survey of 100 extensor alpha motoneurons of the cat
responding to stretch of the gastrocnemius-soleus muscles,
demonstrated that 53% of the motoneurons discharged only
at the beginning of the stretch while the remainder dis-
charged throughout the duration of the stretch. The phasic
alpha motoneurons were also distinguished from the tonic
by their larger axonal spike potentials. Granit, Phillips,

Skoglund and Steg (1957) have shown that activation by the

:euror
higher
(10-2!

Lzed n

l6

crossed extensor and pinna-tW1st reflexes gives the same
discriminatory reSponse between phasic and tonic and moto-
neurons. The phasic motoneurons were shown to exhibit a
higher frequency of discharge (30-60/sec) than the tonic
(10-20/sec). Eccles, Eccles and Lundberg (1958) character-
ized motoneurons by duration of after potentials as well

as by the conduction velocrties of their axons. They
demonstrated that axons which conduct rapidly and have
shorter after-hyperpolarization were character1stics of
neurons innervating white or fast muscles while axons
which displayed slow conduction velocity and a prolonged
after-hyperpolarization innervated red or slow muscles.

It was concluded that, in general, the tonic and phasic
types of motor neurons activate slow and fast muscle re-
spectively. The after—hyperpolarization determines the
frequency of discharge (Eccles, 1936; Eccles, 1953; Pitts,
1943). This is supported by the findings that slowly dis-
charging tonic motor neurons have a longer after-hyperpolar-
ization than the rapidly discharging phasic motor neurons
(Eccles 33 31., 1957a, b; Eccles 33 31, 1958). The tonic
motor neurons innervate slowly contracting red motor units,
which could therefore result in a fused tetanus at a
relatively low frequency of discharge. Phasic motor
neurons have high frequencies of discharge and are matched
to the relatively fast-white muscles which they innervate

and are therefore capable of producing an Optimal tetanic

1?

fusion. Any serious mismatching would result in
ineffic1ency (Eccles 33 31., 1958). This empha51zes the
importance of the neuron in differentiating the fibers
which it innervates into fast or slow types in order to
insure a prOper match. This would also imply a homogeneity
of the motor unit.

The prOperties of motor units in a red muscle,
the soleus, versus a mixed pale muscle, the medial head
of the gastrocnemius were investigated (Wuerker, McPhedran,
and Henneman, 1965; McPhedran, Wuerker, and Henneman, 1965).
An attempt was made to define these types of neurons in
regard to their Speed of contraction which would correspond
to the type A, B, and C fibers of the gastrocnemius as
described histochemically by Stein and Padykula (1962).
The axons supplying type A and type B motor units were
outlined with considerable clarity however the axons of
type C motor units were not clearly defined. The results
however, demonstrated that the average conduction velocity
of the axons supplying the red muscle was considerably less
than that of the axons supplying pale muscle.

Histologically and histochemically the ventral
motor neurons specifically associated with red or white
motor units have not been identified. Physiological
evidence, however, implies a size relationship and a poss-
ible biochemical relationship of the motor neuron to its

motor unit.

In
between the
zsgehnated
amplitudes
also due:
If 1t 18 .
to the 51
ical eva
we may t?
and a rag
(fast) m
In the s
d‘ictlon
0f sma11
seem reg
Innerva-
DEUrons
t0 note

“tog

18

In regard to Size, a direct prOportionality exists
between the axon diameter and the conduction veloc1ty of
myelinated nerve fibers (Hursh, 1939; Rushton, 1951). The
amplitudes of nerve impulses from peripheral axons are
also directly related to their diameter (Gasser, 1941).

If it is assumed that the diameters of axons are related

to the Sizes of their cell bodies, as scattered histolog-
ical eVidence indicates (Henneman, Somjen, Carpenter, 1965)
we may then eXpect that axons displaying a large impulse
and a rapid speed on conduction, characteristic of white
(fast) motor units arise from a large ventral motor neuron.
In the same respect impulses of low aptitude and slow con-
duction velOCity would be eXpected to arise from a neuron
of smaller dimenSions (Somjen 33 al., 1964). It would then
seem reasonable to assume that the white motor units are
innervated by large motor neurons in contrast to small
neurons supplying red motor units. It is also of interest
to note that gamma motor neurons whose axons are small

(1 to 8 microns) innervate intrafusal muscle fibers which
are predominantly red histochemically (Yellin, 1968). Their
tendency to fire continuously even in the resting limb of
an anesthetized animal (Henneman, Somjen, and Carpenter,
1965) would then appear to be a functional expression of
their small Size.

In regard to activity Henneman (1957) demonstrated

that the excitability of ventral motor neurons varied

19

inversely with their size, the reflex threshold of
indiVidual cells increaSing in parallel with the amplitude
of impulses recorded from their axons. In the light of the
preVious discussion it can be concluded that smaller ven-
tral motor neurons, demonstrated to innervate red motor
units, are more susceptible to discharge than are the
larger motor neurons assoc1ated with white motor units.
This concept is in agreement with the findings that slow
muscles are activated in the lowest threshold range of the
postural reflexes while fast muscles are in the upper

range of threshold (Denny-Brown, 1929b). Vrbova (1963)
also demonstrated that the soleus, a red muscle, is almost
continuously activated, according to electromyographic
recordings, whereas a white muscle is activated during
periods of reflex actiVity or voluntary movement. It has
also been Shown that in the post-tetanic potentiated state
(Granit st 31., 1956), extensor motor neurons fall into

two categories in their responses to a constant stretch:
(i) a tonic group which fires for a long duration in a
maintained stretch and (ii) a phasic group which only fires
one or two Spikes on the rising phase of the stretch.
Analysis of Spike Size demonstrate that the tonic group
tends to have smaller spike potentials than the phasic
group and concludes that tonic motor neurons tend to group
themselves among the smaller ventral motor neurons and the
phasic among the larger (Granit 23.3l'r 1956; Granit et 31.,

1957).

20

The results, therefore, of predominantly
physiological studies would suggest not only small neurons
innervating red motor units, but also more active neurons.
Increased functional activity in a neuron is suggestive
of an increased energy metabolism and pOSSible increased
activities of enzymes assoc1ated Wlth the transmission of
impulses along the axons or neuromuscular transmission.

In summary phySiological eVidence demonstrates
that red motor units are innervated by neurons which dis-
play in general the following characteristics with respect
to neurons of white motor units: (1) a lower threshold
of excitability; (2) a long duration of tonic activity in
response to a maintained stretch reflex; (3) smaller Spike
potentials; (4) a slower Speed of conduction; (5) a longer
after-hyperpolarization time; (6) conduct fewer impulses
per second. Analysis of conduction velocity and spike
potentials suggests that these neurons are smaller in size
and their tonic character suggests neurons of increased

functional as well as biochemical activity.

Influence of Exercise

It had not been demonstrated that alterations in
the prOportions of red to white fiber types could occur
‘with.a physical training program, however, quantitative
biochemical studies demonstrated changes in oxidative and

(glycolytic enzymes of exercised muscle (Halloszy, 1967;

21

Peter et at., 1968) which strongly suggested this
possibility. Vrbova (1963) stated that a muscle becomes
slower with continuous use which indicates pOSSible changes
in fiber types With increased functional actiVity. It has
also been suggested that transformations of fiber types
probably occurs throughout life as the result of changing
phySiological demands upon nerve and muscle (Guth gt gt.,
1968). The fact that all muscle fibers are slow at birth
and gradually differentiate into fast types is also suggest-
ive of an influence by functional activity.

A number of investigators have demonstrated mor-
phological changes in ventral motor neurons with exercise
Eve, 1896; Hyden, 1943; Wendt, 1951; Edstrom, 1957).
However, few if any have investigated this problem in
relation to pOSSible alterations in the pr0portions of
red to white fibers in skeletal muscles following long

term exercise programs.

Functional ActiVity and the Motor Neuron

 

Motphological Effect§

 

The literature dealing with the influence of in-
creased functional actiVity upon the morphology of motor
neurons indicates that motor neurons can become altered
.in response to increased activity.

A Wide variety of eXperimental procedures have been
utilized to increase the activity of motor neurons. In-

cluded among these are electrical stimulation, chemical

 

22

irritation, and exerCise all of which have been used at
varying levels of intenSity and duration.

It is believed that the confUSion and contradiction
apparent in this area of investigation is, in part, the
result of diverSity in experimental procedures.

Whether the actiVity of nerve cells is aSSOCiated
with recognizable changes in their structure was first in—
vestigated by Hodge (1889, 1892, 1894). A number of ex-
periments conSisted of stimulating posterior root ganglion
cells through the brachial or sc1atic plexus in frogs or
cats for one to ten hours. A few eXperiments were devoted
to ascertaining if the effects of fatigue could be removed
by varying periods of rest. The ganglia of birds were
also compared before and after a hard days work. The re-
sults of these varied experiments suggested that the
nucleus decreases markedly in Size and becomes dark and
jagged with functional activity. The cell body was re-
ported to become slightly shrunken and vacuolated. It was
also observed that the nerve cells recovered with rest.

A number of early investigators have reported that neurons
enlarge as a result of increased activity whereas Shrink-
age occurs with exhaustion. (Vas, 1892; Mann, 1894; Lugaro,
1895). Others have found that shrinkage is the only effect
of activity with reSpect to volume changes (Hodge, 1889;

Legendre and Pieron, 1908). Negative findings have also

been reported (Eve, 1896; Kocher, 1916; and Hyden, 1943).

lore recentl
response to

:nation of s
-i‘.-.‘e~'ieles, 1‘3-
cdl nuclei

in staining
neurons of e.
fiter a thir
Swelling of

ODStIdCEd 1n

 

 

 

PrOlOnged em
produced on 1‘

Most
and ne‘dron d
Stalnmg int
lty, COP-lpre
of lesl Sub
by Dolley (I
ProcedUreS ‘
on a track,
cell Change
fatigue, an

be ..

23

More recently hypertrOphy of nerve fibers was reported in
response to increased work load produced by surgical elim—
ination of synergistic muscles or the contralateral limb
(Wedeles, 1949). Wendt (1951) demonstrated enlarged nerve
cell nuclei follow1ng Similar denervations. Dimensions
and staining properties were markedly altered in motor
neurons of exerCised gu1nea pigs sacrificed immediately
after a thirty-minute running period (Edstrom, 1957).
Swelling of the cell body, nucleus, and nucleolus was dem-
onstrated in the neurons of these animals. However, a
prolonged exerCise program over a twenty-nine day period
produced only a Slightly enlarged darkly Staining nucleolus.
Most authors in the above discuSSion of exerCise
and neuron dimensions have also reported changes in the
staining intenSitieS of nerve cells with functional activ-
ity. ComprehenSive studies in regard to the stainability
of Nissl substance With increased actiVity were reported
by Dolley (1909a, 1909b, 1911b). Various experimental
procedures were used in these Studies, e.g. dogs running
on a track, administration of drugs, and others. Nerve
cell changes were diVided into Six Stages With activity,
fatigue, and exhaustion and a close relationship was shown
between Nissl substance and the nucleolus. Since these
early reports a number of investigations have been con-
ducted upon the stainability of Nissl substance of neurons

With acute pathological and phySiological states of

24

neuronal activity (Dolley, 1911a, 1913, 1917; Einarson,
1935; Hyden, 1943; Casperson, 1950; Einarson gt gt., 1955;
Vraa-Jensen, 1957; Aleinikova gt gt., 1966).

Einarson (1933) concluded from developmental studies
of neurons that chromatin material was first Visible around
the nucleolus. It then migrated toward the periphery of
the nucleus and diffused gradually through the nuclear
membrane to form the Nissl substance. Continual reforma-
tion could be observed during normal activity of normal
nerve cells as well as during stages of recovery following
artificial stimulation or severe injury to axons. These
concluSions have been corroborated through the comprehen-
sive research of Hyden (1947). Extensive biochemical re-
search has led to a more complete understanding of the
above findings (Einarson, 1957; White gt gt., 1964; Watson,
1965). It is now widely accepted that RNA plays an impor-
tant role in the synthesis of proteins, including enzymes
and that the syntheSis of RNA iS under the control of DNA
of nuclear chaomatin. During develOpment of the neuron
the essential nuclear component (RNA) of Nissl substance
is formed in or near the nucleolus inside the nucleus; it
Inigrates toward the periphery of the nucleus, and then
passes through the nuclear membrane into the cytOplasm.
Subsequently, it is incorporated as an essential constitu-
ent of the complex cytOplasmic nucleoprotein elements,

e.g. the Nissl substance (Binarson, 1933; Casperson, 1950;

25

Einarson, 1957). Nissl substance iS formed not only during
development, which has been Studied by Hyden (1943) and
more recently by Vraa-Jensen (1957) but also in the adult
organism during life. The consumption and reformation are
particularly conSpicuous in conditions of increased activ—
ity, pathological stress and regeneration of the neuron
(Einarson and Krogh, 1955). Likewise, corresponding changes
in protein production in the cell is apparent during growth
and function (Casperson, 1950).

Based upon the works of several investigators
Einarson (1957) described the stainability of Nissl sub-
stance at various levels of functional activity. A
chromoneutral condition represented the staining profile of
nerve cells during a state of normal actiVity. A moderate
chromOphilia (hyperchromasia) characterized cells when
their activity began to increase; moderate chromophobia,
gradually becoming more marked was characteristic of cells
in increasing activity of longer duration. Extreme chrom-
Ophobia represents cells in a state of severe functional
stress, fatigue and exhaustion. ChromOphilia was believed
to be characteristic of cells in a state of prolonged
active inhibition, or cells whose activity was diminished
or abolished for some time. In pathological conditions

extreme chromOphobia may proceed to total dissolution of

the cell.

 

26

Edstrom (1957) was one of the first to compare the
immediate effects of acute exhaustive exercise with that
of a prolonged exercise program. One group of guinea pigs
was exercised to exhaustion by running and immediately
sacrificed, while a second group was exercised for thirty-
two hours in intervals during a twenty-nine day period.
Ventral motor neurons of the acute exercise group showed
a severe chromOphobic reaction in Nissl staining but the
nucleolus remained deeply stained. Neurons of the prolonged
exercise group showed intense staining of Nissl substance
with a light staining nucleolus.

During functional activity it has been shown that
the stainability of neurons is related to a balance between
the rate of RNA production by the nucleus and its rate of
breakdown in the cytOplasm. Alterations in this balance
can result in chromOphobic or chromOphilic staining reac-

tions (Vraa-Jensen, 1956).

Histochemical Effects

 

The role of acetyl choline and acetylcholinesterase
in neuronal transmission and conduction mechanisms has been
subject to numerous investigations since the experiments of
Dale (1914). Pharmacological evidence indicated the role
of AChE to be the removal by hydrolysis of released trans—

mitter agent, acetylcholine, for repeated activity, (Eccles,

1957).

 

27

Since the demonstration of a histochemical technique
for cholinesterase activity (koelle and Friedenwald, 1949)
the distribution of cholinesterases have been studied in a
variety of situations and structures in order to correlate
structure with function. However, these investigations
have not employed the use of physical activity in studying
the cholinesterase distribution in ventral motor neurons.
It is generally accepted that cholinesterases are synthe—
sized in the cell bodies of cholinergic neurons and are
tranSported along the axons to their sites of activity
(Feldberg, 1957). For example, Sawyer (1946) found that
after section of the sciatic nerve in guinea-pigs, the
concentration of acetylcholinesterase in the central stump
increased by about 300% whereas the concentration in the
distal end fell to 40%, indicating a constant production
by the ce-l body and conduction along the axon.

In the mammalian spinal cord granules attached to
the surface of the ventral motor neurons stain deeply with
acetycholinesterase methods and are associated with
synaptic vesicles (Adams, 1965). There is also intense
staining throughout the perikaryon (Roessmann and Friede,
1967).

A number of isozymes of acetylcholinesterase in
nervous tissue have been demonstrated (Bernsohn, Barron,

and Hess, 1962). ,However, the significance of the multiple

nature of acetylcholinesterase has not been illucidated.

 

28

Although the biological functions of pseudocholinesterase
are unknown, they have been implicated in certain lipid
metabolic pathways (Clitherow, Mitchard, and Harper, 1963).
Biochemical studies indicate that butyryl cholinesterase
(BuChE) activity resides predominantly in white matter

(Ord gt gt., 1952; Cavanagh gt gt., 1954). More specif-
ically it has been found to be active in glia cells,
capillary endothelium and smooth muscle of blood vessels
rather than neurons of the central nervous system (Koelle,
1952, 1954; Brightman et a1., 1959).

Acid phosphatase, a hydrolytic enzyme associated
with lysosomes, has been correlated with phagocytosis,
intracellular digestion, secretion, and necrobiosis (DeDuve,
1959). This enzyme has also been associated with the
localization of the complex neuronal lipofuscin pigment
which is actively or passively formed in the cell (Anderson
and Song, 1962). Acid phOSphatase activity has been re-
lated to nucleic acid sites in nerve cells and it has been
suggested to play an important role in an enzyme system
acting upon Nissl substance to release phosphate groups for
cell maintenance and function (LaVelle gt gt., 1954). It
has been shown, for example, that under stressful condi-
tions increased acid phosphatase in neurons is closely
related to the disappearance of stainable Nissl bodies
(Bodian and Mellors, 1944; Smith and Luttrell, 1947).

Acid phOSphatase activity has been reported to be greater

29

at the axon hillock (Smith, 1948). The axon hillock has
been suggested to be a region of "physiological chromato-
lysis" where there is a continuous local breakdown of RNA
(LaVelle gt gt., 1954).

Quantitative biochemical studies have not demon-
strated an increase in acid phosphatase in chromatolytic
neurons. For example, an increase or decrease in the
amount of this enzyme in chromatolytic cells of regenera-
ting axons could not be shown (Fieschi gt_gt., 1959).

Acid phosphatase rich lysomes can become enlarged under
conditions of stress (Strause, 1967). This may provide a
reason for an apparent increase in acid phOSphatase act-
ivity observed in chromatolytic neurons rather than an
actual increase in the quantity of this enzyme.

Dehydrogenase activity has been Shown to be high
in neurons of the central nervous system (Lowry, 1957;
Adams, 1965; Thomas and Pearse, 1961). Malic acid dehydro-
genase is an enzyme of the Krebs' cycle and shows a greater
activity in nerve cell bodies than does glucose-6—phosphate
dehydrogenase, an enzyme of the pentose shunt. A relation-
ship of glucose-6—ph05phate dehydrogenase to Nissl staining
has not been demonstrated in nerve cells, however, the
pentose shunt is an important pathway for the production
of ribose sugars which are incorporated into RNA. In

growing tumors, pyroninOphilia of the rapidly growing cells

is accompanied by increased glucose-6-phosphate

 

3O

dehydrogenase activity (Pearse, 1958; Pearse, 1961).
However, an increase in glucose-6-phosphate dehydrogenase
activity can occur without an increase in RNA production
as it does in the macula densa of kidneys in hypertensive
animals (Pearse, 1958; Hess and Pearse, 1959). The pentose
phosphate shunt also produces reduced triphosphOpyridine
nucleotides required in lipid synthesis.

Few studies have employed the use of exercise to
determine the influence of increased functional activity
upon enzyme histochemistry of the ventral motor neuron.
That dramatic histochemical alterations can occur in
ventral motor neurons of guinea-pigs, forced to run for
30 and 60 minutes and sacrificed immediately, was demon-
strated with adenosine triphOSphatase techniques
(Wawrzyniak, 1963). The dramatic results obtained were
those of a rapid exhaustive type of exercise rather than

a long term exercise program.

MATERIALS AND METHODS

Experimental Animals

 

Seventy-two male Sprague-Dawley rats 100 days of
age were obtained for this investigation. The animals
were immediately housed in sedentary cages for seven days
to provide an environmental adjustment period. Sixty
animals were selected from this group on the basis of
weight. Animals demonstrating extremely high or low body
weights were eliminated. The selected animals were then
placed into groups of three according to similarity of
body weights and each animal of a trio was randomly assigned
to one of three eXperimental groups. The animals were
grouped in this manner in order to minimize extreme diff-
erences in weight as a possible source of variance. The
end result was twenty trios each of which contained a

representative from each of three experimental groups.

Experimental Groups

 

Group A (Sedentary). The animals of this group
were housed in sedentary cages (24 x 18 x 18 cm.) for the
entire treatment period, but were removed from their cages

once each week for body weight determinations.

31

 

32

Group B (Sedentary-forced). This group of rats
was housed in sedentary cages in the same manner as in
Group A. However, they were forced to swim one thirty-
minute period per day with a lead weight equal to three
per cent of their body weights attached to their tails.
Group C (Voluntary-forced). The animals of Group
C were housed in cages of the same dimensions as in Groups
A and B. However, these cages were equipped with voluntary
exercise wheels (35 cm. in diameter and 13 cm. wide) which
allowed the rats to run at will. Revolutions of the volun-
tary wheels were mechanically recorded and activity records
for each animal were taken. In addition, this group was
forced to swim two thirty-minute bouts per day, with a
weight equal to four per cent of their body weights

attached to their tails.

Experimental Treatment

 

Tail Weights

 

The lead weights attached to the rats' tails were
adjusted weekly in accordance with their body weights.
The weights were attached at the tip of their tails rather
than at the base. It was observed that their tails, which
are used to a great extent for balance and support against
the sides of the tank, were immobilized to a greater
extent by placing the weight at the tip. This treatment

forced the use of their legs to a much greater extent in

33

maintaining balance and swimming. The weights were
attached by means of miniature plastic Clothespins which
facilitated attachment and removal. They also provided a
secure attachment of the weight during the swimming period.
Trauma to the tip of the tail by the Clothespins was pre-

vented by protection with adhesive tape.

Swimming

 

The sedentary-forced grOUp (B) of animals swam
thirty minutes each day between the hours of nine o'clock
a.m. and twelve o'clock noon. This treatment was continued
Six days a week over a period of 52 days.

The voluntary-forced group (C) received the same
experimental treatment as Group B with an additional thirty-
minute swim per day between the hours of five and eight p.m.
A minimum of five hours of rest therefore intervened be-
tween exercise bouts.

Previous experience has Shown that a training
period of approximately 50 days is sufficient to produce
alterations in skeletal muscle of rats and that continued
training for longer periods of time becomes detrimental to
the animal.

In order to facilitate sacrifice and tissue pro-
cessing procedures two triplicates (6 animals) were sacri—
ficed on ten consecutive days. It was therefore necessary

to initiate into the swimming program an additional two

34

triplicates on ten consecutive days in a sequence
corresponding to the sequence of sacrifice. This permitted
the duration of treatment to be constant for all animals.

The rats were swum in individual cylindrical tanks
eleven inches in diameter and thirty inches deep. Follow-
ing each swim the animals were dried with towels and re-
turned to their respective cages. During a swimming period
the rats were observed closely. If an animal remained sub-
merged for more than ten seconds it was removed from the
tank for a brief rest period and the weight was removed
its tail. The animal was then returned to the tank with
no weight attached to its tail.

The sedentary-forced group of animals demonstrated
considerably more difficulty in tolerating the swimming
program. Four rats died during the swimming treatment,
three of which belonged to the sedentary-forced (B) group.
The fourth rat belonged to the voluntary-forced group (C).
The exercise program imposed upon the sedentary-forced
group (B), therefore, did not appear to be well suited for
adaptation by these animals to the training program. The
increased difficulty in swimming displayed by these animals

continued throughout the exercise program.

Environment and Food

 

The ambient temperature of the animal quarters

ranged from 21° C to 25° C. The water temperature of the

35

swimming tanks ranged from 32° C to 34° C. The water in
the tanks was renewed at each swim period. The animal
rooms were automatically regulated to maintain twleve hours
of light (eight a.m. to eight p.m.) and twelve hours of
darkness (eight p.m. to eight a.m.).

All animals were fed ad libitum with commercial

block feed. (Wayne)

Sacrifice Procedures

 

Six animals per day (two triplicates) were sacri-
ficed on ten consecutive days. On each day following their
final treatment each rat from the group of six animals was
anesthetized intraperitoneally with two ccs. of 1% Pento-
barbital. The lumbar intumescence of the spinal cord was
removed and trimmed of its spinal nerves in two to five
minutes. The removed portion of the cord was then divided
at the fifth lumbar segment. The upper portion was fixed
in ten per cent formalin for histological procedures and
the distal portion was rapidly frozen for histochemical
analyses. All sections were taken from the region of the
4th, 5th and 6th lumbar segments which contribute to the

formation of the sciatic nerve in the rat (Green, 1955).

36

Histochemical Procedures

 

Freezing

 

The spinal cord block was mounted, in gum traga-
canth, on a metal chuck. Only the chuck was submerged
into iSOpentane which was cooled to a viscous fluid by
liquid nitrogen. Complete submersion was observed to crack
this tissue. Approximately forty-five seconds were re-
quired to completely freeze the block of tissue when only
the chuck was submerged. No more than one to two minutes
elapsed between the time of removal and the transformation

of the tissue to the frozen state.

Cutting

Immediately after freezing, the block was placed
into an Ames Lab Tek cryostat in which the temperature of
the tissue was allowed to equilibrate to that of the cryo-
stat (between minus fifteen and minus twenty degrees centi-
grade). Sections of cord used for cholinesterase and
pseudocholinesterase procedures were cut at twenty micra.
Those for all other procedures were sectioned at ten micra.
These thicknesses were observed to give the best results
in preliminary staining trials of the spinal cord. Two to
five sections, not less than 20 microns apart, were cut
from each block for histochemical procedures. The sections
‘were placed on cover Slips and fan dried while incubation

:media were being prepared.

37

Incubation

Freshly prepared incubation media were prepared
daily. All sections to be stained each day for a specific
enzyme were incubated together in the same medium for the
same length of time. Comparisons and evaluations were
made only within the group of animals processed on the
same day. Incubation times for cholinesterase and pseudo-
cholinesterase were five hours, but those for all other
enzymes were thirty minutes. All sections were mounted on
slides with glycerine jelly.

Control sections were incubated periodically for
each procedure to determine the specificity of the reac-
tions. In some cases the substrate was excluded from the
medium while in others the coenzymes, diphosphoPyridine
nucleotide (DPN) or triphosphOpyridine nucleotide (TPN),
were deleted in procedures which normally require them for
satisfactory reactions. Control sections for cholinester-
ases were also preincubated for one hour in Er600 (di-
ethyl-P-nitroPhenyl phOSphate) at 10-7 molar concentration.
E-600 has been shown to be a powerful cholinesterase

inhibitor at the above concentration (Pearse, 1961).
Histochemical Techniques

Cholinesterase (CHE)

Demonstration of cholinesterase activity in ventral

Imotor neurons was accomplished by the acetylthiocholine

38

method (Koelle and Friedenwald, 1949). This procedure
employs acetylthiocholine iodine as the substrate and is
hydrolyzed by both acetycholinesterase (AChE) and pseudo-
cholinesterase at a pH of 6. Parallel sections were
incubated in the same basic medium but with butyrylthio-
choline iodine as the substituted substrate, which is
hydrolyzed specifically by pseudocholinesterase. In both
reactions the c0pper and sulfate present in the medium
combine with the released thiocholine to produce a c0pper
thiocholine sulfate complex which is converted to c0pper
sulfide by passage of the section through dilute ammonium
sulfide. The brown deposits of c0pper sulfide mark the
sites of cholinesterase activity. The difference in stain-
ing intensity between the two procedures provides an

estimation of acetylcholinesterase (AChE) activity.

Acid Phosphatase (A.Pase)

 

The procedure used to demonstrate acid phosphatase
activity was that of Barka (1960). The method is based
upon the hydrolysis of alpha naphyl phosphate (substrate)
by acid phosphatase to liberate naphthol at an Optimal pH
of 5. This compound then couples with one or more azo
groups on each molecule of freshly diazotized pararosani-
lin to form an azo dye produce which is highly insoluble
and resistant to ordinary organic solvents (Davis and

Ornstein, 1959). Barka's hexazonium pararosanilin

39

technique has been demonstrated to be particularly suitable
for the demonstration of acid phOSphatase in neural tissue

(Anderson and Song, 1962).

Malic Acid Dehydrogenase

 

The procedure for malic acid dehydrogenase con-
sisted of incubation of the tissue in a medium composed of
one ml. of one molar malic acid, ten mg. of diphosphopyrid-
ine nucleotide (DPN), fifteen mg. of nitro blue tetrazolium
salt (NBT), and 10 m1. of 0.2M Tris buffer at a pH of 7.4.
The final pH was adjusted to 7.4 with ten normal sodium
hydroxide.

The oxidation of malic acid by malic acid dehydro-
genase converts DPN to its reduced form. In the oxidation
of reduced DPN the terazolium salt (NBT) becomes reduced
and is converted to an insoluble colored formazan product

which marks the Sites of MDH activity.

Glucose-6-phosphate dehydrogenase
(G-B-PD)

 

Glucose-6-phosphate dehydrogenase catalyzes the
reaction from glucose-6-phOSphate to 6-phosphogluconolac-
tone, a reaction characteristic of the pentose phosphate
shunt. This reaction is accompanied by the reduction of
the coenzyme triphosphoPyridine nucleotide (TPN). The

reoxidation of this compound results in a reduction of the

. o 4' up.

 

4O

tetrazolium salt (NBT) to a colored, insoluble, formazan
product.
Both dehydrogenase techniques are those of Hess

‘gt gt. (1959) as modified by Barka gt gt. (1963).

Histological Techniques

 

The proximal portion of spinal cord was fixed in
10% formalin and embedded in paraffin. Sections were cut
at seven microns and stained for Nissl substance with
Luxol fast blue and neutral red (Lockard and Reers, 1962)
and with toluidine blue 0 (Stenram, 1954). All sections
were carried through the staining procedures together and
were stained and destained for the same lengths of time.
A subjective indication of the degree of chromophilia in

the three groups was obtained from these sections.

Measurements of Neurons

 

Photographs of right and left ventral horns from
each animal were taken at lOOX magnification using a Zeiss
photomicroscoPe. Negatives obtained from this procedure
were mounted in slide holders and the images of ventral
horns were projected onto Sixteen by twenty-four inch
sheets of paper. The projected images of the neuron cell
body, nucleus, and nucleolus was then outlined., Areas of
the traced cellular features were measured with a compen-
sating planimeter. The average of three consecutive read—

ings was taken as the final area for each cell feature.

41

A stage micrometer was also photographed and the negative
projected under the same conditions to determine the mag
nification of the traced neurons for conversion to square
microns. Approximately 2000 ventral motor neurons were
measured without knowledge of the experimental group from
which they came. The mean areas for each experimental

group were then computed and converted to square microns.
Evaluation and Analysis

Histochemical

 

Histochemically the motor neurons, in two to five
sections of spinal cord per animal, were graded into dark,
medium and light staining cells according to intensity of
reaction. All ratings were made without knowledge of the
treatment groups. The percentages of dark, medium and
light staining cells were computed for each animal as well
as the mean percentages for each treatment group (Appendix
.A). Acid phOSphatase reactions permitted grading of motor
neurons into only dark and light categories. Approximately
2000 ventral motor neurons were rated for each enzyme
studied.

The resulting data were analyzed using a one—way
analysis of variance (Guenther, 1964). Differences in the
:percentages of dark, medium and light staining cells were
examined among the three treatment groups. When the F—

:ratio was significant the Duncan multiple range test was

42

used to determine which means differed (Bliss, 1967).
One-way analysis of variance performed on the
percentages of dark, medium and light staining cells in
these treatment groups involves an inherent interdependence
of data. For example, determining percentage of dark
staining cells of one group limits the percentages of
medium and light staining cells of that group. However,

there is independence of data within each test.

Reliabilitprata

 

One month following the initial evaluation a second
determination of the percentage of dark staining neurons
was performed on approximately one third of the animals
selected randomly from each group (Appendix A). A linear
correlation was employed to determine the correlation be-
tween the percentages of dark staining neurons obtained on
the first and second determinations.

In addition to the evaluation stated above, the
animals of each group were rated from one through four
based upon an overall impression of enzyme activity in the
motor neurons of the sections examined (Appendix A). This
evaluation served to indicate whether or not changes in
the percentages of dark, medium and light neurons were
reflected in an overall subjective impression of their
staining intensity. It was apparent that a change in

enzyme activity could occur in all ventral motor neurons

43

without alteration in percentages of dark, medium and light
cells. These subjective ratings were tested using a con-

tingency chi-square to determine significant trends.

Measurement Data

 

Data involving area measurements (Appendix C) was
analyzed using a one-way analysis of variance (Guenther,
1964). Differences were examined in regard to areas of
cell bodies, nuclei, and nucleoli among the three experi—
mental groups. When the F-ratio was Significant the Duncan
multiple range test was utilized to determinewhich means
differed (Bliss, 1967). One-way analysis of variance
tables for both histochemical and area data are recorded

in Appendix B.

RESULTS

Histochemical Results

 

Cholinesterase

 

The ventral motor neurons of all experimental
groups showed an intense staining reaction at the periphery
of the cell bodies (Figs. 1, 2, 3). The staining reaction
throughout the remaining cell body was less intense but
uniform. Less cholinesterase activity was observed in the
nucleus than in the surrounding perikaryon, however, the
nucleolus exhibited a very strong reaction (Figs. 13, 14).

The neurons of the ventral horn differed from one
another in staining intensity of their cytOplasm. On the
basis of these differences the neurons were categorized
into dark, medium and light staining cells. The staining
reaction of the perikaryon was the most obviously affected
feature of neurons in the treatment groups for all enzymes
studied.

The ventral horns of the sedentary group (A) of
(animals demonstrated a preponderance of moderate to heavy
staining cells (Fig. 1). The nuclei showed little enzyme

activity while the nucleoli were heavily stained.

44

45

The sedentary-forced group (B) showed a marked
decrease in cholinesterase activity in the perikaryon as
well as in the nucleoli of these neurons. The intense
cholinesterase activity at the periphery of the cells
appeared to be little affected in these animals and there-
fore appeared even more pronounced in cells showing de-
creased activity in the perikaryon (Fig. 2).

The ventral motor neurons of the voluntary—forced
group demonstrated staining reactions similar to those of
the sedentary group with the exception that the dark
staining neurons displayed an even greater intensity in
the perikaryon and nucleolus than those of the sedentary
group.

Table 1 demonstrates the changes in percentages
of dark, medium and light staining cells in the three ex-
perimental groups. Each group representing the evaluation
of six to seven hundred ventral motor neurons. The lower
portion of Table 1 shows the F-ratios obtained in each
category, from a one way analysis of variance and secondly
which groups differed Significantly using the Duncan
multiple range test.

The most marked alteration in activity occurred in
the sedentary-forced (B) group (Table l). The percentages
of dark and medium staining cells were significantly lower
than in the sedentary group while the percentage of light

intensity cells was significantly higher than in the

46

sedentary group. No significant difference in the
proportions of cell intensity categories was observed
between the sedentary and voluntary-forced groups. How-
ever, in subjective evaluations the neurons and neuropil
of the voluntary-forced group appeared more intensely
stained than the sedentary group which is reflected in a
frequency distribution of subjective ratings Shown in

Table 2.

Table l
CHOLINESTERASE

Mean percentages of dark, medium and light stain-
ing neurons in three eXperimental groups

 

 

 

 

 

 

% Dark % Medium % Light
Group Cells Cells Cells
B - Bed.
forced 8.02 35.84 56.13
C --vol.
forced 49.23 35.01 16.35
F .05 Between Groups
% D 35.75 s* A - B s*
% M 11.67 s* B - C s*
% L I 56.00 s* A - C ns*

 

s* - significant
ns* - non-Significant

47

Subjectively the motor neurons from each animal
were collectively given a rating from one through four
based upon an overall impression of their staining inten—
sity (Appendix A). The intensities were rated as follows:

1. light 3. intense
2. moderate 4. very intense

The following table represents the distribution of

these ratings in the three treatment groups.

Table 2

Frequency distribution of subjective ratings
for cholinesterase activity in treatment groups

 

 

 

Intensity Group A Group B Group C
Rating Sed. Sed.-Forced Vol.-Forced
l 1 9 0
2 6 2 l
3 5 l 9
4 0 0 2

 

The subjective data for cholinesterase activity
does not satisfy the basic assumption for a contingency—
chi-square test (not more than 20% of the cells may have
a frequency of less than five).. The frequency of distri-
bution, however, is in an expected direction based upon

the percentage data presented previously.

48

Pseudocholinesterase activity in the ventral horns
and ventral motor neurons in all three groups was observed
to be very slight. Distinguishing individual neurons with-
in the equally light staining neurOpil was difficult
(Fig. 19). The paucity of pseudocholinesterase activity
in the ventral motor neurons suggested that the intense
enzyme activity, described in the eXperimental groups of
animals as cholinesterase activity, was predominantly that
of acetylcholinesterase. The reactivity demonstrated by
acetylthiocholine procedures in this study will, therefore,

be referred to as acetylcholinesterase (AChE) activity.

Glucose-G-Phosphate Dehydrogenase

(a-g-PD)

The activity of G-6-PD was observed to be consider-
ably stronger in the ventral motor neurons than in the
surrounding neurOpil in all treatment groups. The intensity
of staining throughout the perikaryon was characteristically
uniform. Absence of activity in the nucleus and nucleolus
was evident in all neurons. Different staining intensities
between the neurons permitted their classification into
three intensity groups (dark, medium and light). The mean
percentages of cells in each category per animal is given
in Appendix A.

Table 3 demonstrates the mean percentages of dark,
medium, and light staining neurons representing G-6-PD

activity in each treatment group. Five to six hundred

neurons were evaluated for each group.

49

The F-ratio and

significance between groups are recorded at the bottom of

the table.

Table 3

GLUCOSE - 6 - PHOSPHATE DEHYDROGENASE

Mean percentages of dark, medium and light staining
neurons in three eXperimental groups

 

 

 

 

 

 

 

% Dark % Medium % Light
Group Cells Cells Cells
A - sed. 34.94 50.87 14.16
B - 88d.
forced 17.19 30.52 59.60
C "' V01.
forced 26.92 54.15 16.93
F .05 Between Grqpps
% D 5.20 s* A — B s*
% M 7.84 S* B - C S*
% L 30.49 s* A - C ns*

 

*s - significant

*ns - non—significant

The overall picture observed with the percentage

data (Table 3) is very similar to that observed in Table

l for cholinesterase.

The sedentary-forced group again

50

exhibits a marked decrease in the percentages of dark and
medium staining neurons and an increased percentage of
light staining cells. The voluntary-forced animals do not
differ significantly from the sedentary animals. The
differences in G-6-PD activity of the three groups is
illustrated in Figs. 4, 5 and 6.

Subjectively, a rating (one through four) was
eassigned to the neurons of each animal based upon an over—
all impression of reactivity (Appendix A). The intensities
were rated in a manner Similar to that used for cholinester-
ase:

1. light 3. intense
2. moderate 4. very intense

Table 4 below illustrates the frequency distribu-
tion of the assigned ratings for G-6-PD in the three ex-
perimental groups. The distribution is similar to that

in Table 2 for cholinesterase.

Table 4

Frequency distribution of subjective ratings
for G-6-PD activity in treatment groups

 

 

 

Intensity Group A Group B Group C
Rating Sed. Sed.-Forced Vol.-Forced
l 1 5
2 7 8
3 8 1 7
4 l 0 23

 

Significant at .05 level.

51

Collapsing this data by combining the intensity
ratings 1 with 2 and 3 with 4 permitted a contingency chi-
square to be used to determine significance. A Signifi-
cant difference was determined between the distribution of
ratings in the treatment groups. The sedentary—forced
group demonstrates a significant decrease in G-6-PD activity.

A slight increase in G-6-PD activity in the volun-
tary-forced group is indicated by the frequency distribu-

tions of the sedentary and voluntary-forced groups.

Acid Phosphatase (A.Pase)

 

Acid phosPhatase activity was observed in all
ventral motor neurons examined. Generally, these showed
discrete staining throughout their cytOplasm. Increased
staining at specific sites in the cytOplasm was not a con—
sistent feature of the sedentary and voluntary—forced groups
but many cells of the sedentary-forced group demonstrated
an increased perinuclear staining (Fig. 8). Nuclear and
nucleolar activity was absent in all neurons.

The variation in staining intensity between neurons
was small but permitted their classification into two in-
tensity categories designated dark and light. Approximately
eight hundred neurons were evaluated and the mean percent-
ages of dark and light neurons were obtained for each
group. The results of this evaluation are given in Table

5. The F-ratios and the significance between groups are

52

indicated in the lower part of the table.

Table 5
ACID PHOSPHATASE

Mean percentages of dark and light staining
cells in three eXperimental groups

 

 

 

 

 

 

% Dark % Light
Group Cells Cells
B - sed.
forced 40.43 59.51
C - V01.
forced 23.71 76.27
F .05 Between Groups
% D 5.17 s* A - B s*
% L 4.16 s* B - C s*
A - C ns*

 

*s - significant
*ns - non-significant

The results of this evaluation Show an increased
acid phosphatase activity in the neurons of the sedentary-
forced group of animals. This is in contrast to the
results of CHE and G-6-PD reactivity observed in this
group. However, it is again the sedentary-forced group

which has demonstrated the most marked alteration in

53

enzyme activity. The voluntary-forced group Shows no
significant change in the percentages of dark and light
neurons from the sedentary animals.

The reactivity of acid phosphatase was subjectively
rated as in the evaluation for CHE and G-6—PD. Intensity
ratings of one through three were employed:

1. light 2. moderate 3. heavy
The frequency distribution within the three experimental

groups is shown in Table 6.

Table 6

Frequency distribution of subjective ratings
for A.Pase activity in treatment groups

 

 

 

Intensity Group A Group B Group C
Rating Sed. Sed.-Forced Vol.-Forced
1 8 l 8
2 8 9 8
3 3 5 1

 

Significant at .05 level

The distribution was collapsed by combining intens-
ity ratings 2 and 3 in order to utilize a contingency chi-
square. A significant difference was obtained among the
frequency distributions of the treatment groups. The
results of this evaluation are in agreement with the per-

centage data (Table 5) showing a significant increase in

54

A.Pase activity in the sedentary-forced group with no
significant difference between the sedentary and voluntary-
forced groups.

Malic Acid Dehydrogenase
(MDH)

Malic acid dehydrogenase activity was observed to
be uniform throughout the cytoplasm. Many neurons of the
voluntary-forced group of animals showed an increased
staining intensity at or near axonal and dendritic origins.
Nuclei and nucleoli demonstrated no enzyme activity (Fig.
15).

Table 7 illustrates the percentages of dark,
medium and light staining cells obtained in each experi-
mental group of animals and the F-ratio obtained for each
intensity category. No Significant differences were ob-
tained between the eXperimental groups.

Table 8 illustrates the frequency distribution of
subjective ratings assigned to the neurons of each animal.

The overall intensities were rated as follows:

1. light 3. intense
2. moderate 4. very intense

55

Table 7
MALIC ACI D DEHYDROGENASE

Mean percentages of dark, medium and light
staining cells in three eXperimental groups

 

 

 

 

 

 

% Dark % Medium % Light
Group Cells Cells Cells
A - sed. 12.94 51.58 34.44
B " sed.
forced 20.67 53.14 26.16
C - vol.
forced 30.29 50.95 23.90
E .05
% D 1.72 ns*
% M .07 ns*
% L 1.11 ns*

 

*ns - non-Significant

Table 8

Frequency distribution of subjective ratings for
MDH activity in treatment groups

 

 

 

Intensity Group A Group B Group C
Ratings Sed. Sed.-Forced Vol.-Forced
1 9 4 2
2 8 7 5
3 3 5 9
4 0 l 3

 

Significant at .05 level

56

A contingency chi-square performed on this data,
after collapsing l with 2 and 3 with 4 intensity categories,
showed a significant difference between the distributions
in the treatment groups. A trend toward greater activity
is observed in groups B and C. The subjective ratings
assigned to the general reactivity in these groups may have
been influenced by the increased MDH activity evident in
the neuropil immediately surrounding the neurons of the
exercise groups. On the other hand, a trend toward in-
creased MDG activity in neurons of both sedentary-forced
and voluntary-forced groups could conceivably occur without
an accompanying change in the relative prOportions of dark,

medium and light staining neurons in these groups.

Reliability Data

The percentage of dark staining neurons in each
treatment group was determined in two separate evaluations.
This was done for each enzyme and on approximately one-
third of the animals of each group (Appendix A). The
correlation between the two sets of data for each enzyme

is recorded in Table 9.

57

Table 9

Correlation from reliability data

 

 

 

Enzymes Correlation
CHE 80
G-6-PD 93
A.Pase 87
MDH 98

 

Morpholggical Results

 

Nissl Staining

 

Luxolfast blue and toluidine blue staining of
ventral motor neurons showed almost identical results.
Neurons of the sedentary group of animals demonstrated
moderate to heavy staining with an intensely staining
nucleolus. Those of the sedentary-forced group demon-
strated a distinct chromophobic reaction with an indistinct
and lightly staining nucleolus. The Nissl substance was
less coarse than that of the sedentary group. Neurons of
the voluntary-forced group of animals showed a more intense
staining of Nissl substance which generally obscured the
nucleus (Figs. 10, ll, 12, 17, 18). A deeply stained
nucleolus, however, could be observed in these cells. The
intensity of staining in the voluntary-forced group was
generally more intense than that observed in the sedentary

group of animals.

58

Measurements of Neurons

 

The mean areas of ventral motor neurons, their
nuclei and nucleoli in each of the treatment groups are
given in Table 10. The mean areas for each cell feature
per animal is recorded in Appendix C.

Approximately two thousand ventral motor neurons
were measured. The F-ratios for each cell feature are

given at the bottom of the table.

Table 10

Mean areas of ventral motor neurons in
Square Microns

 

 

 

Cell Group A Group B Group C
Feature Sed. Sed.-Forced Vol.-Forced
Cell Body 271.2398 244.2521 234.8018
Nucleus 63.5164 66.6968 59.2456
Nucleolus 12.1762 17.5374 10.9041

 

 

Between Groups

 

 

F .05 (Nucleoli)
Cell Body 1.88 ns* A - B s*
Nucleus .50 ns* B - C 8*
Nucleolus 5.55 s* A — C ns*

 

*s - Significant
*ns — non—significant

59

The results recorded in Table 10 do not Show a
significant difference between the mean areas of cell bodies
in the three treatment groups. The mean for group B, how-
ever, is less than that of A and the mean area of group C
is less than that of group B. Although the differences
are not significant at the 0.05 level, a decrease in the
mean areas of groups B and C is apparent, and was the only
feature that paralleled the increasing levels of functional
activity.

The differences between the mean areas of the
nuclei are not significant at the 0.05 level. The nucleo-
lus, however, shows a marked increase in area in the
sedentary-forced group of animals, while the nucleolar
area of the voluntary-forced group of animals does not

differ significantly from that of the sedentary group.

Body Weights

 

The final body weights for each animal are recorded
in Appendix C. The mean body weights for each group are
given in Table 11. A significant decrease in body weights
occurred in the sedentary-forced group and an even greater
decrease in the voluntary-forced group.

The percent correlation between final body weights

and mean neuronal areas is very low in all three groups.

60

Table 11

Mean body weights and per cent correlation between
final body weights and mean neuronal areas

 

 

 

 

Group A Group B Group C
Sed. Sed.-Forced Vol.-Forced
Mean Body
Weight* 424.30 371.52 333.63
% Correlation .07 31 13
* A>B>C - significant at .05 level

DISCUSSION

With one exception the intracellular distribution
of AChE described in this study is in agreement with other
histochemical and quantitative biochemical studies.
Giacobini (1959) analyzing microdissected parts of motor
neurons found no activity in the nucleoli of the cells.
Intense staining of the nucleolus for AChE in neurons of
all eXperimental groups was evident in this study and is
in agreement with intense activity of AChE in ventral
motor neuron nucleoli reported by Roessmann and Friede
(1967). The reason for the disparity between the quantita-
tive findings of Giacobini and those of histochemical
studies is not clear.

The neurons displayed an intense reactivity at the
periphery of the cell bodies, with a lesser activity in the
perikaryon. This was consistent with the internal and ex-
ternal fraction of neuronal AChE (with respect to the cell
membrane) described by Koelle gt_gt., (1956) and Koelle
(1962). It has been demonstrated that anti-cholinesterase
agents inhibit only the external fraction in sympathetic
ganglion cells and was therefore termed "functional AChE."
The internal fraction, probably associated with the endo-

plasmic reticulum (Fukuda and Koelle, 1959; Toschi, 1959)

61

62

could be inactivated without immediate apparent effects

and was called "reserve AChE" (McIsaac and Koelle, 1959).

Sedentary-Forced Group

 

The decreased AChE activity exhibited in the
sedentary-forced group of animals was apparently the result
of a reduction in the "reserve AChE." The decreased act-
ivity was probably the result of diminished production of
the enzyme by the cell or a rapid depletion due to trans-
port along the axon to the motor end plates with increased
muscular activity. In contrast, the cell bodies of the
voluntary-forced group, in which the exercise program was
more severe, exhibited increased AChE activity. A dimin-
ished production, rather than depletion by utilization,
would therefore appear to be an important factor in the
decreased AChE activity in the neurons of the sedentary-
forced group of animals.

Nissl substance of the cell bodies of the sedentary-
forced group exhibited a chromophobic reaction with the
nucleus containing an enlarged pale staining nucleolus.
During continued functional activity RNA is used up, the
breakdown exceeding synthesis which presents a typical
chromoPhobic appearance of the nerve cells (Vraa-Jensen,
1957). However, chromOphobia may also indicate a condition
of increased protein production. Regenerating neurons

demonstrate an increase in organic cell material during a

63

period when the amount of RNA remains unchanged and the
neurons present a typical chromatolytic appearance
(Brattgard, Edstrom and Hyden, 1957). The chromatalytic
appearance suggesting a transformation of Nissl substance
to a more active form rather than a reduction in amount.
The reduced AChE and G-6-PD activity in the sedentary-
forced group of this study would suggest a diminished
amount of functioning RNA.

Edstrom (1957) studied the stainability of Nissl
substance in the ventral motor neurons of guinea pigs ex-
hausted after a single running exercise period. He also
studied the neurons of animals after a prolonged exercise
program. The neurons of Edstrom's exhausted animals Showed
a chrom0phobic reaction in their Nissl staining very similar
to those of the sedentary-forced group of the present study
with the exception that the nucleoli of the exhausted
animals were deeply staining and apparently unaffected.

The nucleoli of the sedentary-forced group in this study
were.markedly enlarged and pale staining. That the neurons
of exhausted animals resembled very much the neurons of the
sedentary-forced group in this study also suggests a poorly
adapted state of the cells to the increased activity.

An increased acid phosphatase activity was observed
in the neurons of the sedentary-forced group. This finding
substantiates the close relationship of acid phOSphatase

and the breakdown of Nissl substance (LaVelle gt_gl., 1954),

64

since the neurons of this group Show a chromOphobic response.
The decreased levels of glucose-6-phosphate dehydrogenase
activity is suggestive of a decreased RNA production. This
enzyme belongs to the pentose phosphate shunt which is an
important pathway for the production of ribose sugars,
necessary for the synthesis of RNA. No significant change
was observed in malic acid dehydrogenase activity in this
group of animals from those of the sedentary group. Shapot
(1957) suggested that energy produced in a nerve cell is
utilized for structural metabolism (renewal of cell struc—
tures and protein production) and for specialized functional
metabolism. It is possible that during increased functional
activity most of the energy produced by the cell would be
used to satisfy the functional demands of the neuron at the
expense of protein synthesis at least until a metabolic

adaptation to the increased activity could take place.

Voluntary-Forced Group

 

Although the animals of this group were subjected
to a more severe and more continuous exercise program for
the same time period, their motor neurons showed a metab-
olic response quite different from those of the sedentary-
forced group. The ventral motor neurons demonstrated an
overall increase in AChE activity above that of the seden-
tary animals as well as increased g1ucose-6-phosphate

dehydrogenase activity. A chromoPhilic response was

65

observed in the Nissl substance of these cells as well as
an intensely staining nucleus and nucleolus. Diminished
acid phosphatase activity corresponding to the chromOphilic
response was also observed.

The neurons of guinea pigs (Edstrom, 1957) sub-
jected to a prolonged exercise treatment by treadmill
running (exercised thirty-two hours in intervals during
twenty-nine days) showed a staining of Nissl substance
comparable to that of sedentary animals. The nucleoli of
these neurons were enlarged and pale staining. The volun-
tary—forced group of the present study showed a more
intense staining of Nissl substance than their sedentary
control counterparts and the nucleoli were deeply stained.
The increased baSOphilia in the cyt0plasm of these ventral
motor neurons as well as the enzymatic changes are con-
sistent with neurons adapted to an increased level of
activity.

It is the author's Opinion that the Opposite
metabolic responses Obtained between the sedentary-forced
and voluntary—forced groups is a result of the different
exercise programs to which these animals were subjected.
The sedentary-forced group was vigorously exercised for a
thirty-minute period per day and then forced to rest for
the remaining twenty-three and one half hours. It would
appear that each exercise period begins an adaptive phase

by the neuron which then regresses during the resting

66

stage, preventing complete metabolic adaptation by the
neuron.

The voluntary-forced group of animals, however,
were exercised for two thirty-minute periods per day, and
exercised at will in cages equipped with voluntary exercise
wheels as demonstrated by activity records obtained from
voluntary wheel revolution data. Under this more continu-
ous type of activity the neurons were able to adapt even
though the exercise program was more vigorous than that
of the sedentary-forced group. In short, the neurons adapt
with greater difficulty to the type of exercise programs
imposed upon the sedentary-forced (B) group of animals,
than that imposed upon the voluntary-forced (C) group.

Although the muscle fibers of the plantaris
(Edgerton, 1968) showed a moderate adaptation by a Signif-
icant increase in the prOportion of red to white fibers,
the neurons demonstrated a poorly adapted state by their
enzymatic activity and chromophilic reactions. The seden-
tary-forced group of animals also demonstrated increasing
difficulty in COping with their exercise program. This
was demonstrated by their inability to keep up with the
swimming pace for a thirty-minute period even though they
swam with less weight (3% Of their body weights) than the
voluntary-forced group (4% of their body weights). It is
also interesting to note that three of the four animals

that died while swimming belonged to this sedentary-forced

67

group. The increased difficulty in swimming displayed by
these animals was reflected in the ventral motor neurons
rather than the skeletal msucle.

The exercise program to which the sedentary-forced
group was subjected appeared to be detrimental to the ven-
tral motor neurons as was reflected in the exercising
capabilities of the animals. These results should be con-
sidered in treatment of muscle myOpathies with exercise
programs and in the design of eXperiments related to the
effects of exercise treatments on pathological conditions

such as muscular dystrOphy.

Relationships to Muscular Results

Edgerton (1968) demonstrated an increase in the
proportion of red to white muscle fibers in the plantaris
muscle of these same eXperimental animals. A significant
increase in this pr0portion occurred in the sedentary-
forced group with an even greater increase in the voluntary-
forced group. The results of a number of physiological
studies concerned with neuromuscular relationships (see
review of pertinent literature) suggest that ventral motor
neurons innervating red motor units are smaller in size and
functionally more active. In regard to size changes, the
mean area of ventral motor neurons decreased in the seden-
tary-forced group and decreased to an even greater extent

in the voluntary-forced group. Although these changes were

68

not significant at the 0.05 level, there was a consistent
decrease in mean areas with increasing levels Of activity.
This was the only morphological feature studied which
paralleled the increasing levels of functional activity

and prOportions of red to white fiber types as reported by
Edgerton (1968). In judging the biological significance

of this data, it must be remembered that the neurons occupied
the region of the fourth, fifth and sixth lumbar segments Of
the spinal cord and innervated a large number of muscles in
addition to the plantaris. Certainly, all of the muscles

of the extremity would not be eXpected to be exercised to
the same degree nor to demonstrate the same alterations as
shown in the plantaris. However, results from the plantaris
muscle suggest Similar trends in other mixed skeletal mus-
cle though not necessarily to the same degree. Muscles
which were already predominantly red to begin with would

not be expected to show this trend. For example, no alter—
ations in the pr0portions of intermediate to red fibers

were Observed in the soleus muscles of the exercised groups
(Edgerton, 1968).

Another factor which Should be considered is the
size of the motor unit. It has been shown, for example,
that the average motor unit Of the medial gastrocnemius of
man contains approximately 1400 muscle fibers (Fernstein

gt gl., 1955). An alteration of ten motor units from white

to red represents a change in the intensity of staining of

69

14,000 muscle fibers in the medial gastrocnemius.

Assuming a corresponding change in neuronal areas, the

same alteration would be reflected in the areas of only
ten motor neurons of the 4th, 5th, and 6th lumbar segments.
It therefore seems reasonable that significant alterations
of motor units should be more readily reflected in the
proportions of red to white fibers of specific muscle
rather than in the areas of neurons in the lumbar spinal
cord.

Although the mean areas of neurons in the eXperi-
mental groups suggest a decreasing size of the neuron with
increasing levels of activity and increasing prOportionS
of red to white fibers in the plantaris muscle, definite
conclusions in this regard await more specific biochemical
and morphological studies of neurons innervating specific
muscles.

The ventral motor neurons innervating red motor
units have been shown to be much more active in terms of
excitability, susceptibility to discharge, and have been
demonstrated to fire almost continuously in the resting
extremity of an anesthetized animal (Wuerker, gt gt., 1965;
McPhedran gt gl., 1965; Henneman gt gt., 1965). It might,
therefore, be anticipated that an exercise program which
increased the prOportions of red muscle fiber types in
skeletal muscle would be reflected in an increased pro-

portion of neurons demonstrating intense activity of

70

enzymes related to energy production and function of the
neuron. This concept was not immediately reflected in

the histochemical findings of the sedentary-forced group
of animals. Indeed AChE, an enzyme related to the func-
tion of alpha motor neurons, and g1ucose-6—phosphate
dehydrogenase, related to energy metabolism and to ribose
production for RNA synthesis, demonstrated a marked de-
crease in activity. The prOportion of cells showing in-
tense activity of these enzymes diminished greatly, and a
general chromOphobia was observed in the neurons of this
exercised group. A possible eXplanation for these unex—
pected findings might be found in the fact that the motor
neurons were functioning at an increased level of activity
during the exercise periods, but were unable to adapt to
the brief bouts of forced exercise separated by long periods
of complete rest. The decreased prOportions of enzymat-
ically active cells was an uneXpected manifestation of
chronically increased activity and, in terms of functional
activity, was consistent with the increased prOportions of
red to white muscle fiber types as indicated by the find-
ings of Edgerton (1968).

Ventral motor neurons of the voluntary-forced group
demonstrated increased prOportions of active cells showing
increased enzyme activity corresponding to a prolonged
increased functioning and an accompanying increased meta—

bolism.

71

Limitations of This Study and Suggestions
For Future Studies

 

 

The functional significance of cell size in the
nervous system has been emphasized and more specifically
that of the ventral motor neurons has been related to the
varied types of motor units in skeletal muscle (Henneman
gt gt., 1965). This is based upon physiological evidence
only in that the spike potential and speed of conduction
are proportional to axonal diameter and the assumption that
axonal diameter is directly related to the Size of the
neuron from which it arises. It is the author's opinion
that investigations on the morphology of ventral motor
neurons supplying red versus white Skeletal muscle would
be extremely valuable to further research in this area.

It might then be possible to substantiate with morpholog-
ical evidence the conclusions drawn from physiological
data. Studies such as this would also provide a precise
localization of neurons in the spinal cord which innervate
specific muscles and would provide a more sound basis for
correlating changes in ventral motor neurons with changes
in muscle fiber types under various pathological and
physiological conditions.

Similarily, a thorough histochemical investigation
Of neurons as related to the muscle fiber types they supply
would be extremely valuable for future studies in this area
of research as well as autoradiographic studies on RNA and

protein turnover in these cells.

72

There is an absence of histochemical and
morphological evidence for the existence of "red and white
alpha motor neurons." An attempt, therefore, to relate
changes in ventral motor neurons to alterations in the
prOportions of muscle fiber types is somewhat premature.
However, the results of this study suggests changes in
.ventral motor neurons which are consistent with the current
concepts of red and white motor units and the increased
prOportion of red to white fibers in the skeletal muscle
of chronically exercised animals. It is the author's pre-
sent ambition to investigate Specifically the morphological
and histochemical features Of neurons supplying predomin-
antly red or predominantly white muscles.

In considering the results of exercise on ventral
motor neurons it is quite apparent that different intens-
ities as well as different types of exercise programs may
vary the morphological and histochemical features of motor
neurons and that more studies of this kind would add
greatly to an understanding of the effects of chronic
exercise.

It became apparent, during the course of this study,
that physiological analysis of the motor nerves, in regard
to speed of conduction and after-hyperpolarization, would
have provided a wider SCOpe of information on which to

interpret morphological and histochemical results.

SUMMARY AND CONCLUSIONS

Summary

Ventral motor neurons of sedentary and chronically
exercised adult male rats were studied histochemically
using CHE, BuChE, A.Pase, G-6-PD and MDH techniques. The
mean areas of ventral motor neurons and the stainability
of Nissl substance were also considered.

Sixty male Sprague—Dawley rats, 100 days of age,
were placed into three groups. The control group (A) was
housed in sedentary cages. The animals of a sedentary-
forced group (B) were placed into sedentary cages but were
exercised by swimming thirty minutes per day with a weight
equalling three per cent of their body weights attached to
the animals' tails. A voluntary-forced group (C) was
housed in cages equipped with voluntary exercise wheels
and received two thirty-minute forced exercise swims per
day. The animals of this group swam with weights equal to
four per cent of their body weights. The duration of the
exercise treatment was fifty-two days.

Fresh frozen sections from the fourth, fifth and
sixth lumbar segments were obtained for CHE, BuChE, A.Pase,
MDH, and G-6-PD techniques. Formalin fixed, paraffin

embedded sections were also taken for toluidine blue and

73

74

luxol fast blue neutral red techniques.

Histochemically the motor neurons were graded
according to intensity and the percentages of neurons fall-
ing into each intensity category were recorded for each
animal of the eXperimental groups. A subjective rating of
overall intensity of activity in the neurons of each animal
was also recorded. Measurements of neuron areas were Ob-
tained using a compensating planimeter upon projected
negatives of ventral horns.

Approximately 2000 ventral motor neurons were
evaluated for each enzyme and for area measurements with-
out knowledge of the experimental groups from which they
came.

The sedentary-forced group of animals demonstrated
a diminished CHE, G-6-PD activities, and all animals of
this group showed some degree of chromOphobia in their cell
bodies. An increased acid phOSphatase activity was ob-
served in these neurons. The animals of this group also
exhibited increasing difficulty in OOping with the exercise
program.

The voluntary-forced group Showed intense activity
Of CHE and G-6-PD in their neurons along with a deminished
acid phosphatase activity. A chromophilic reaction was
also observed in the neurons of this group.

BuChE, an enzyme Of unknown function, Showed very
little activity in the ventral motor neurons of all three

groups.

75

The mean areas of ventral motor neurons for the
treatment groups decreased with increased levels of
activity, however, the differences observed were not
significant at the 0.05 level. The nucleolus of the sed-
entary—forced group exhibited a significant increase in
size while that of the voluntary-forced group showed no
significant change from the sedentary control animals.

The results of the present study are discussed in
regard to the nature of the exercise programs and in the
light of alterations in the proportions of red to white

fiber types in skeletal muscle with chronic exercise.

Conclusions

 

The following conclusions were drawn of the results
of this study.

1. That an exercise program consisting of short
bouts of swimming, to which the sedentary-forced group of
animals was subjected, presents a metabolic picture of
neurons poorly adapted to the increased activity of the
exercise periods.

2. Rats subjected to such an exercise program
exhibit increasing difficulty in swimming a thirty-minute
period and therefore become poorly adapted to this treat-
ment.

3. Metabolically, neurons and the skeletal muscles

they innervate do not appear to adjust simultaneously to an

76

exercise program such as imposed upon the sedentary-forced
animals.

4. A more continuous and intensive exercise treat-
ment results in neurons which appear to be metabolically
better equipped to meet the demands of increased functional
activity. Animals subjected to this treatment were also
better adapted physically to the swimming periods. rfifii

5. That the increased proportions of cells Show-

ing intense enzyme activity and the decrease in the mean

 

areas of cells in the voluntary-forced group of animals 5~1
appears to be consistent with the increased proportions of v,
red to white fiber types in exercised muscle, suggested
by the results of Edgerton (1968). It is also consistent
with the physiologically derived data that motor neurons
supplying red fibers are smaller in Size and functionally
more active.
6. That another similar study should be done, but
prefaced by studies on the histochemistry and morphology
of neurons specifically related to red and white muscles
and their specific location in the Spinal cord of the rat.
From such studies more definite conclusions could be drawn
concerning the adaptability of the motor neuron, as an
integral part of the motor unit, to physiological and

pathological changes in functional activity.

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84

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89

FIGURE 1

Cholinesterase activity in ventral motor neurons (arrows)
of a sedentary animal. Increased activity is seen at the

periphery of the cells and intense activity throughout
the perikaryon. (340 X).

FIGURE 2

Cholinesterase activity predominant in the neurons Of the
sedentary-forced group of animals. Diminished activity

is seen in the perikaryon while intense activity remains
at the periphery. (340 X).

FIGURE 3

Ventral motor neurons of the voluntary-forced group of
animals incubated for cholinesterase. Intense reactivity

in both the perikaryon and periphery of the cell bodies
is noted. (340 X).

PLATE I

 

90

 

91

FIGURE 4

G-6-PD activity in the ventral motor neurons of a

sedentary animal. Dark and medium staining cells are
prevalent. (290 X).

FIGURE 5

Activity of G-6-PD in the motor neurons of the voluntary-
forced group showing increased numbers of light staining
cells. One dark (upper left corner) and three light
neurons (middle left) are seen in this figure. (290 X).

FIGURE 6

G-6PD activity in the ventral horn of a voluntary-forced
animal. Increased numbers of dark and medium intensity
cells are evident. Absence of nuclear and nucleolar
activity was evident in cells of all groups. (290 X).

PLATE II

 

92

93

FIGURE 7

Acid phosphatase activity in two neurons of the sedentary
group of animals. Nuclear and nucleolar activity was
absent from the cells of all groups. (320 X).

 

 

; , FIGURE 8

Increased acid phOSphatase activity in neurons of the
sedentary-forced group Of animals. Increased staining
intensity surrounding the nucleus is very evident in the
neuron at the middle left margin of this figure. (320 X).

FIGURE 9

Diminished acid phosphatase activity in motor neurons of
the voluntary-forced group of animals. Moderate and light
staining cells are seen in this figure. (320 X).

PLATE III

 

 

95

FIGURE 10

Nissl staining in ventral motor neurons of a sedentary
animal. A deeply staining nucleolus and moderately

stained Nissl substance were evident in these neurons.
(toluidine blue - 320 X).

FIGURE 11

Chromophobic ventral motor neurons in the sedentary-forced
group of animals. These neurons represent the most chrom-
Ophobic reactions Observed, however, a paler staining

Nissl substance and a light staining nucleolus were con-

stant features in neurons of this group. (toluidine blue -
320 X).

FIGURE 12

A severe chromophilic reaction in neurons of the voluntary-
forced group of animals. Deeply stained nucleoli and
intense staining of Nissl substance, often obscuring

nucleus, are Observed in these neurons. (toluidine blue -
320 X).

 

96

_
* 9:81»

 

97

FIGURE 13

CHE activity in a medium staining cell from a sedentary-
forced animal. Nucleolar activity is seen and moderate
staining throughout the perikaryon. (710 X).

FIGURE 14

CHE activity in a ventral motor neuron of a voluntary-
forced animal. Intense nucleolar staining is clearly
demonstrated. Less activity is observed in the nucleus
with greater activity in the remainder of the cell body.
(710 X).

FIGURE 15

MDH activity in two motor neurons from the sedentary
group of animals. Absence of nuclear and nucleolar
activity and intense cytoplasmic activity was Observed
in all treatment groups. (460 X).

FIGURE 16

Acid phosphatase in a neuron from the sedentary group.

An increased activity is observed at the base of a
neuronal process. A complete absence of nuclear staining
is seen. (520 X).

FIGURE 17

Toluidine blue stained neuron from the sedentary-forced
group. A mild chromOphobic reaction and a pale staining
nucleolus can be seen. A distinct nuclear outline is
also evident. (540 X).

FIGURE 18

Toluidine blue stained neuron from the voluntary-forced
group. A somewhat obscured nucleus and intensely staining
Nissl substance and nucleolus can be seen. (540 X).

FIGURE 19

BuChE in the ventral horn from a voluntary-forced animal.
A uniformly slight distribution is observed throughout
the ventral horn with increased activity in blook vessels.

   

98

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0.0M M.MH
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HOEHCO 30mm mo A.m.mv OCHumH 0>HuooOnsm cam HOEHOO HOQ

mHHOO OcHsHmum Adv uanH Osm HOV xumw mo usmo mom

 

mm¢8<mmmomm QHUN

ozo޴

106

MO H OM OH
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OO NN ON O

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0.0H 0.00 0.0 O N O.MO O.NH MM M 0.0H N.HO O.MH ON
0.0H 0.00 N.M OO H 0.00 0.0 ,NN H H.NO 0.00 0.0 OH
M.MN 0.00 M.N NM N O.NO M.ON OO N M.MM 0.00 H.O MH
O.NM 0.00 M.MH OO H 0.00 0.0 OO H M.OO .O.MO 0.0 ON
O.MH 0.00 0.0N MM N O.MO 0.0 OM N O.MN H.N 0.0 OM
0.0 0.0N 0.00 N N 0.00 N.OH MO. H M.O 0.00 M.ON OM
0.0 0.0N 0.0M OO M 0.0M 0.0N OO H 0.00 0.0N 0.0 M

0.00 H.OO 0.0 OO M 0.0M O.MO ON H O.HM N.MH 0.0 NH
0.0M M.HO 0.0H OH H H.MN 0.0 M M M.OH. N.OO 0.0N NO
0.0 0.00 M.MM HO N N.MO 0.0 OM M M.ON 0.00 MMOH OO
0.0N H.OO M.OH HM M 0.00 N.MH ON H 0.0N 0.00 0.0N OH
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OM.OH O0.0H O0.0 O0.0 O0.0 O0.0
mmfizmwomawmma madmmmommlOlmmOUDHU
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mamuo> umuHO mnu OH Oochuno mHHoo mchHmum MHOO mo usoo mom

 

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109

O0.00
MO.HH
ON.OH
OO.H

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OO.MO

O0.00
O0.0
OM.MH
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MM.N
OO.H
ON.OH
OO.HO
OO.MM

O0.0
O0.0
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O0.0N

 

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OH.N
ON.MH
OH.O
OO.NO
OO.MH

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OH.O
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N0.00
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APPENDIX B

110

 

111

CHOLINESTERASE

 

One way analysis of variance tables.
Per cent dark staining cells:

Source of

 

VafIance St. SS M88
Among levels 2 10,207.4422 5,103.7211
Within levels 33 4,710.2879 142.7359
Total 35 14,917.7301

Per cent medium staining cells:

Source of

 

Variance St. SS M88
Among levels 2 2,089.7027 1,044.8513
Within levels 33 2,955.1256 89.5492
Total 35 5,044.8283

Per cent light staining cells:

Source of

 

Variance St. SS M88
Among levels 2 12,320.4341 6,160.2170
Within levels 33 3,630.0694 110.0021

Total 35 15,950.5035

I”!

35.75

I"!

11.67

[’21

56.00

 

112

GLUCOSE- 6 -PHOSPHATE DEHYDROGENASE

 

One way analysis of variance tables.

Per cent dark staining cells:

Source of

 

Variance St. SS
Among levels 2 4,541.4018
Within levels 45 19,635.3543
Total 47 24,176.7561

Per cent medium staining cells:

Source of

 

Variance St. SS
Among levels 2 4,887.9158
Within levels 45 14,032.9766
Total 47 18,920.8924

Percent light staining cells:

Source of

 

Variance St. SS
Among levels 2 19,313.7756
Within levels 45 14,252.1292

Total 47 33,565.9048

MSS

2,270.7009
436.3412

MSS

2,443.9579
311.8439

MSS

9,656.8878
316.7139

I”!

30.49

 

113

ACI D PHOSPHATASE

 

One way analysis of variance tables

Per cent dark staining cells:

Source of

 

Variance St. SS MSS
Among levels 2 3,188.1121 1,594.0560
Within levels 31 9,870.8276 308.4633
Total 33 13,058.9397

Per cent light staining cells:

Source of

 

Variance St. SS MSS
Among levels 2 2,623.4490 1,311.7245
Within levels 31 10,096.6931 315.5216
Total 33 12,720.1421

Body Weight and Treatment Groups

One way analysis of variance

Source of

 

Variance St. SS MSS
Among levels 2 80,975.12 40,487.56
Within levels 53 53,234.87 1,004.43

Total 55 134,209.99

In;

40.30

 

114

MALIC ACID DEHYDROGENASE

 

One way analysis of variance tables

Per cent dark staining cells:

Source of

 

Variance St. SS
Among levels 2 1,586.4628
Within levels 53 24,355.8290
Total 55 25,942.2918

Per cent medium staining cells:

Source of

 

Variance St. SS
Among levels 2 45,2163
Within levels 53 17,815.6582
Total 55 17,860.8745

Per cent light staining cells:

Source of

 

Variance St. SS
Among levels 2 1,199.2786
Within levels 53 28,592.2361

Total 55 29,791.5147

MSS

793.2314
459.5439

MSS

22.6081
336.1440

MSS

599.6393
539.4761

I)

[’11

 

I”!

.07

I”!

1.11

115

VENTRAL MOTOR NEURON AREAS

One way analysis of variance tables

Cell body:

Source of

 

Variance - St. SS MSS
Among levels 2 0.0155 0.0077
Within levels 51 0.2119 0.0041
Total 53 0.2274

Nucleus:

Source of

 

Variance St. SS MSS
Among levels 2 0.0005 0.0002
Within levels 51 0.0206 0.0004
Total 53 0.0211

Nucleolus:

Source of

 

Variance St. SS MSS
Among levels 2 632.0284 316.0142
Within levels 48 2,729.1034 56.8563

Total 50 3,361.1318

 

APPENDIX C

 

 

116

117

O0.0H O0.0H M

ONHO. MOHO.

 

OMHO.
OOHO. OMHO.
ONHO.
OHHO. OOHO.
OMOO. OONO.
OOHO. OOHO.
ONHO. OMHO.
MOOO. OONO.
NMHO. OONO.
OOOO. NMHO.
OOOO. HMOO.
OHHO. NMHO.
OOOO. OOHO.
OOOO. ONHO.
OOHO. HOHO.
MOOO. OHHO.
OONO.
MMHO. ONHO.
U m
mDHomHODZ

 

r Mfllr
H.NH ON.OO O0.00 NO.MO
OMHO. NOOO. OMOO. OOOO.

 

ODOMU mmm mz<m2

 

OMHO. OOOO. MOOO.

ONOO. OOOO. OOOO.
HNNO. ONMO. MOOO. MNOO.
HOOO. OOOO. OOOO. NNOO.
OMHO. OOOO. OOOO. OHOO.
HNHO. OOMO. OOOO. OOOO.
OOHO. OOOO. MOOO. OOOO.
HHHO. OMOO. OMOH. NOOO.
HNHO. NHOO. ONOO. OOOO.
OOHO. NOOO. ONOO. MHOO.
OOHO. OHOO. HMMO. OOOO.
OOHO. OOOO. OMOO. OHOO.
OOOO. MOMO. ONOO. HMOO.
NOHO. OOOO. OOOO. OOOO.
HOHOO NONO. OOMO. OOOO.
HMHO. HOOO. HOOO. ONOO.
MONO. HHOO. NOOO. OONH.
OOHO. MOOO. NOOO. MOOO.

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118

 

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