Motor unit properties following cross-reinnervation of cat triceps surae muscles

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Title:
Motor unit properties following cross-reinnervation of cat triceps surae muscles
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x, 196 leaves : ill. ; 29 cm.
Language:
English
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Foehring, Robert C., 1955-
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Subjects

Subjects / Keywords:
Motor Neurons   ( mesh )
Muscle Contraction   ( mesh )
Cats   ( mesh )
Neuromuscular Junction   ( mesh )
Neuroscience thesis Ph.D   ( mesh )
Dissertations, Academic -- Neuroscience -- UF   ( mesh )
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non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1985.
Bibliography:
Bibliography: leaves 181-195.
Statement of Responsibility:
by Robert C. Foehring.
General Note:
Typescript.
General Note:
Vita.

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University of Florida
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Full Text












MOTOR UNIT PROPERTIES FOLLOWING CROSS-REINNERVATION
OF CAT TRICEPS SURAE MUSCLES.












By

ROBERT C. FOEHRING


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA


1985










ACKNOWLEDGEMENTS


I would like to thank the members of my supervisory committee, Drs.

John Munson, Don Stehouwer, George Sypert and Floyd Thompson, for their

advice and interest throughout the course of my dissertation project.

Dr. Janet Zengel was extremely helpful at several stages of this

project. Expert technical assistance was provided by William Ireland,

C.J. Thomas and Audrey Kalehua.

I am especially grateful to Drs. John Munson and George Sypert, who

have been excellent teachers, admirable role models, and great company

for the past few years.

Deb and Christen deserve special thanks for putting up with the life

of a graduate student, and making this all seem worthwhile.

Finally, I thank all those responsible for the microcomputer.

I have been supported as a graduate research associate on an

National Institute for Neurological and Communicative Diseases and

Stroke (NINCDS) grant to Dr. John Munson. The W.L. Gore corporation

donated Gore-Tex for use as nerve-sleeves.


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TABLE OF CONTENTS


ACKNOWLEDGEMENTS ....... .................................... ......

LIST OF TABLES ..... ............................. ... ........ ......

LIST OF FIGURES...................................................

ABSTRACT ............... ....... ..................................

CHAPTERS

I. INTRODUCTION .................... ..........................

II. METHODS................... ........................... ..... .

Initial Surgery .........................................
Acute Experiments. .............................. .....
Whole Muscle Twitch.........................................
Motor Unit Studies.. ............ ................. ..........
Muscle Histochemistry .......................................
Statistical Considerations................ .................

III. NORMAL MG AND LONG-TERM REINNERVATION OF M3 ................

Introduction................... ......... .. ......... ......
Results ........................ ...........................
Discussion .... ......................................

IV. AXOTOMY AND THE TIME COURSE OF SELF-REINNERVATION OF MS....

Introduction................ .. .... .......................
Results. ....... ..... ............................... ......
Discussion............................ ..... .......... ......

V. PROPERTIES OF NORMAL LG AND SOLEUS.........................

Introduction...............................................
Results............... .................. ...............
Discussion ............ ................. ...................

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ii

v

vii

ix



1

6

6
8
9
9
11
13

15

15
18
43

50

50
52
78

90

90
91
111










VI. CROSS-REINNERVATION OF LO AND SOLEUS MUSCLES BY MG MOTOR
NERVE: SELECTIVITY OF REINNERVATION AND MOTONEURON
INFLUENCE ON MUSCLE........................................ 117

Introduction ............................................. 117
Results.................................................. 119
Discussion................................................... 139

VII. CROSS-REINNERVATION OF LG AND SOLEUS MUSCLES BY MG MOTOR
NERVE: MUSCLE INFLUENCE UPON MOTONEURONS.................... 149

Introduction .................... ..... ................... ... 149
Results............................... ..................... 151
Discussion................. ..... ....... ..... ............ 167

VIII. CONCLUSIONS .... ........................... .oo .... ....... 175

LITERATURE CITED ................ ...................... ......... 181

BIOGRAPHICAL SKETCH.......,........................................ 196


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LIST OF TABLES

TABLE PAGE

3-1 Whole Muscle Properties: Normal and Nine Month
Self-Reinnervated MG. ...... ............................. 19

3-2 Muscle Unit Contractile Properties (Long-Caged vs.
Non-Caged; Normal vs. Nine Month Self-Reinnervated)..... 22

3-3 Motoneuron Electrical Properties (Long-Caged vs.
Non-Caged; Normal vs. Nine Month Self-Reinnervated)..... 27

3-4 Differences Between Motor Unit Types: Normal vs. Nine
Month Self-Reinnervated MG.............................. 28

3-5 Data for Non-Contracts (Nine Month Self-Reinnervation
Model) ................ ... ..................... ....... 36

3-6 Motor Unit Types, Muscle Fiber Types and Innervation
Ratios (Normal and Nine Month Self-Reinnervated)........ 38

4-1 Whole Muscle Properties: Time Course of
Self-Reinnervation...................................... 53

4-2 Motoneuron Electrical Properties: Axotomy and Time
Course of Self-Reinnervation.............................. 56

4-3 Low-Re Motoneuron Electrical Properties: Contracts vs.
Non-Contract....................... ........... ..... 66

4-4 Motoneuron Electrical Properties of Non-Contracts:
Self-Reinnervation ................................... 66

4-5 Muscle Unit Contractile Properties: Time Course of
Self-Reinnervation ..................................... 68

4-6 Significance of Differences Between Motor Unit Types for
Med-Re Motor Units....................................... 71

4-7 Motor Unit Types, Muscle Fiber Types and Innervation
Ratios: Time Course of Self-Reinnervation.............. 76

5-1 Whole Muscle Properties: Normal MG, LG and Soleus....... 92

5-2 Percent Muscle Fiber Types by Innervation Compartment;
Normal LG.............................. ........ ....... 92

5-3 Muscle Unit Contractile Properties: Normal MG, LG and
Soleus................ ..................... ..... ..... 98
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5-4 Muscle Fiber Areas By Innervation Compartment: Normal LO 100

5-5 Motoneuron Electrical Properties: Normal MG, LG and
Soleus.................................................. 100

5-6 Significance of Differences Between Motor Unit Types for
Normal MG ............. ............ .. ............... 101

5-7 Motor Unit Types, Muscle Fiber Types and Innervation
Ratios for Normal M3, LG and Soleus............. .... 110

6-1 Whole Muscle Properties: Cross-Reinnervation ........... 120

6-2 Percent Motor Unit Types................... ............ 120

6-3 Muscle Unit Contractile Properties: Normal vs.
Cross-Reinnervated ... ...... ........................... 127

6-4 Percent Muscle Fiber Types By Innervation Compartment
(Long-X)........ o ... .......... ...... ................ 131

6-5 Percent Muscle Fiber Types: Normal and
Cross-Reinnervated .................................. 137

6-6 Significance of Differences Between Motor Unit Types
(Long-X)......................... ... ................... 137

6-7 Significance of Differences Between Motor Unit Types
(Med-X) .............. ... ... ..... ......... ..... ...... 137

6-8 Motor Unit Types, Muscle Fiber Types and Innervation
Ratios (Long-X) ........................... ...... .... 140

7-1 Data for Non-Contracts (Long-X)........................ 152

7-2 Motoneuron Electrical Properties: Cross-Reinnervation... 154

7-3 Significance of Differences Between Motor Unit Types
(Long-X)................................................ 164

7-4 Significance of Differences Between Motor Unit Types
(Med-X).......... ...................................... 164


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LIST OF FIGURES


FIGURE AGE

2-1 Cross-Reinnervation surgery............................ 7

3-1 Frequency histograms for muscle unit contractile
properties: normal and long self-reinnervated......... 24

3-2 Frequency histograms for motoneuron electrical
properties: normal and long self-reinnervated......... 30

3-3 Relationships between AHP half-decay time and axonal
conduction velocity: normal and long self-reinnervated
MG ................................................ 32

3-4 Relationships between rheobase and input resistance:
normal and long self-reinnervated MG................... 34

3-5 Photomicrographs of glycogen-depleted motor unit........ 42

4-1 Frequency histograms for motoneuron rheobase and input
resistance: normal vs. axotomy........................ 59

4-2 Frequency histograms for motoneuron AHP half-decay time
and axonal conduction velocity: normal vs. axotomy.... 61

4-3 Relationships between rheobase and input resistance:
axotomy, low-re and med-re............................ 63

4-4 Relationships between AHP half-decay time and axonal
conduction velocity: axotomy, low-re and med-re....... 65

4-5 Time course of recovery of motoneuron electrical
properties: self-reinnervation........................ 90

5-1 Frequency histograms for normal LG and soleus: muscle
unit contractile properties ........................... 96

5-2 Location of 31 motor units in normal LG................. 98

5-3 Frequency histograms for normal LG and soleus:
motoneuron properties................................... 105

5-4 Relationships between rheobase and input resistance
in normal LG and soleus motoneurons.................. 108

5-5 Relationships between AHP half-decay time and axonal
conduction velocity in normal LG and soleus
motoneurons...... ................................... 110

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5-6 Photomicrographs of normal LG and soleus muscle
histochemistry ........................ ................. 114

6-1 Frequency histograms for normal LG and longX-LG
muscle unit contractile properties.................... 131

6-2 Frequency histograms for normal soleus and longX
soleus muscle unit contractile properties.............. 135

7-1 Frequency histograms for electrical properties of
self-regenerated MG motoneurons, and MG motoneurons
which innervated longX-LG.................. ............ 158

7-2 Relationship between AHP half-decay time and axonal
conduction velocity in MG motoneurons which innervated
LG or soleus.......... ...... ......... ..... .. ....... 160

7-3 Relationships between rheobase and input resistance in
M3 motoneurons which innervated LG or soleus.......... 162

7-4 Frequency histograms for electrical properties of
self-regenerated MG motoneurons, and MH motoneurons
which innervated longX-soleus............. ............ 164

7-5 Time course of recovery of motoneuron electrical
properties: self and cross-reinnervation............... 174


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Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy


MOTOR UNIT PROPERTIES FOLLOWING CROSS-REINNERVATION
OF CAT TRICEPS SURAE MUSCLES

By

Robert C. Foehring

August, 1985

Chairman: Dr. John B. Munson
Major Department: Neuroscience

This study showed several aspects of the relationships between

cat triceps surae alpha-motoneurons, and the muscle fibers they

innervate. Relationships between motoneuron electrical properties and

muscle unit contractile properties were investigated for normal medial

gastrocnemius (MG), lateral gastrocnemius (LG) and soleus motor units.

The relationships between motoneuron and muscle fibers were similar in

quality and strength for normal LG and soleus and for normal MS.

Surgical section of the MG nerve, followed by re-anastomosis to MG nerve

(self-reinnervation), or to the combined LG-soleus nerve

(cross-reinnervation), was employed to examine motoneuron influence on

muscle phenotype, and effects of muscle on the expression of motoneuron

electrical properties. Functional connection to muscle was a necessary

condition for expression of normal, mature motoneuron electrical

properties. Long-term self-reinnervation resulted in complete recovery

of motoneuron electrical properties, muscle unit contractile properties,

and the relationships between them. Reinnervation of a foreign muscle

(LG), with similar original muscle fiber type composition to MG, also

resulted in complete recovery of these parameters. Muscle fibers
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of soleus, an almost purely slow muscle, resisted the influence of Ho

motoneurons. Since MO motoneurons failed to convert soleus muscle

fibers, MG motoneuron properties were altered, thus maintaining a close

relationship between motoneuron electrical type and muscle unit

contractile type. This relationship was of similar strength to that

between normal and also self-reinnervated MG motoneurons and M3 muscle

fibers, but of different quality In MG motoneurons which innervated

soleus muscle, incomplete recovery from axotomy is a possible

explanation for the altered values for motoneuron rheobase, input

resistance and axonal conduction velocity. Afterhyperpolarization

potential (AHP) half-decay time of MG motoneurons which innervated

soleus muscle was longer than in MG motoneurons which innervated MH

muscle, whereas axotomy resulted in no change in mean AHP half-decay

time. Incomplete recovery cannot explain this finding. Thus type of

muscle innervated influences expression of motoneuron electrical

properties. A retrogradely transported chemical message from muscle is a

likely mediating mechanism.
















CHAPTER I
INTRODUCTION



The mammalian neuromuscular system has been an important model

system for study of how cells influence the expression of

properties of other cells. In particular, the expression of

muscle phenotype has been shown to be under strong neural influence.

Buller, Eccles, and Eccles surgically re-routed the nerve from the

'slow' soleus muscle into the 'fast' flexor hallucis longus muscle

(FHL), and vice versa, in young cats (cross-reinnervation; Buller et al.

1960). This study illustrated two major points. First, the type of

nerve influenced the contractile speed of the whole muscle; the 'fast'

FHL muscle became slow under soleus nerve influence, and the 'slow'

soleus muscle became faster, when innervated by FHL nerve. Second, the

conversion of 'fast' muscle by a 'slow' nerve was more complete than the

reverse. These initial observations have been repeated several times,

and at finer levels of resolution, including single motor unit studies

(see references cited in CHAPTER III). Collectively, these studies

provide strong evidence for neural regulation of muscle phenotype.

The electrical activity pattern of cross-reinnervated muscles is

that of the muscle originally innervated by the nerve (Sperry, 1945;

Cohen, 1978; Brinkman et al. 1983; Mulkey, 1983; O'Donovan et al., in

press ). That is, the activity pattern of the nerve is unchanged when

innervating a foreign muscle. This observation has led most workers to

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regard motoneuron properties as uninfluenced by the particular muscle

innervated (e.g. Gordon, 1983). A similar conclusion was reached by Kuno

et al. (1974b), who measured action potential overshoot, axonal

conduction velocity, and afterhyperpolarization (AHP) duration in soleus

motoneurons, up to five months after self- or cross-reinnervation

of flexor digitorum longus muscle (FDL).

Relatively little is known, however, about the expression of

motoneuron electrical properties, and the relationships between them,

after reinnervation of a foreign muscle. A complication in early studies

was that motor units within a muscle were regarded as a single,

homogeneous population. Thus Kuno et al. regarded medial gastrocnemius

(M3) or FDL motoneurons as uniformly 'fast', and soleus motoneurons as

uniformly 'slow'. MG and FDL, although predominately 'fast', are

actually 'mixed' muscles, containing several types of motor unit, and

soleus has occasional fast units (reviewed in Burke, 1981). Changes may

be differential between motor unit types, following reinnervation.

This study utilizes the the cat triceps surae neuromuscular system

as an experimental model of motor unit properties. The triceps surae

includes the soleus, MG and lateral gastrocnemius muscles (LG), all of

which are ankle extensors. The motor unit is defined as an alpha

motoneuron and the muscle fibers it innervates, the muscle portion alone

being referred to as the muscle unit (Burke, 1981). MG motor units can

be classified into four types, based on contractile characteristics of

the muscle unit (Burke, 1981). Motor units can be divided into fast or

slow on the basis of twitch time-to-peak, or the tension profile in an

unfused tetanus ('sag'; Burke et al. 1973). The fast units can be









subdivided on the basis of resistance to fatigue, into fast-fatiguable

(type FF), fast-fatigue-resistant (FR), and fast with intermediate

fatigue-resistance (FI). All slow, type S units are highly

fatigue-resistant. Normal MH contains approximately 45% type FF, 5% type

FI, 25% type FR and 25% type S motor units (Burke, 1981; Fleshman et al.

1981). Normal soleus contains nearly 100% type S units (Burke, 1981).

Normal LG has not previously been studied independent from MG, but is

considered to be similar to MG in motor unit types (from muscle

histochemistry; Ariano et al. 1973; see also Hamant, 1977).

Motoneuron electrical properties have been shown to vary according

to motor unit type, in MG (Fleshman et al. 1981; Zengel et al. 1985).

Parameters investigated include axonal conduction velocity, AHP

half-decay time (related to the ability of the cell to fire

repetitively), rheobase (an inverse indicator of cell excitability), and

input resistance (influenced by cell size and geometry, as well as

specific membrane conductances). Zengel et al. (1985) devised a method

to classify motoneurons on the basis of their electrical

characteristics. This scheme was found to predict motor unit type,

determined independently by contractile properties, with greater than

90% accuracy. In light of the close relationship evident between

motoneuron electrical properties and muscle unit contractile properties

in normal MG, it is of interest whether this is a general property of

normal motor units of other triceps surae muscles, and whether

motoneurons exhibit plasticity in response to experimental manipulation.

In this study, experimental alteration of the innervation of

triceps surae muscles is used to investigate several aspects of the









relationship between motoneuron and muscle. MH motoneurons were isolated

by intracellular methods, to allow recording of motoneuron electrical

properties. Intracellular current injection was employed to activate the

muscle unit to measure contractile responses. In addition, whole muscle

contractile properties and muscle histochemical properties were

documented. Acute experiments were performed on normal, unoperated-on

animals, animals whose MG nerve had been sectioned (prior to

reinnervation of muscle), and animals whose MG and LG-soleus (combined

nerve) nerves had been sectioned, and allowed to reinnervate either the

original muscle (self-reinnervation), or the opposite muscles)

(cross-reinnervation). Acute experiments for reinnervated muscles were

performed at various times after the initial surgery (from three weeks

to ten months). All studies herein that involved surgical manipulation,

were analyzed for MG motoneurons only.

The questions addressed in this study are as follows:

1) Are the relationships between motoneuron electrical properties

and muscle unit contractile properties of similar quality and strength

in LG and soleus to that observed in MG, (CHAPTER V)?

2) Is functional connection to muscle required for expression of

normal, mature motoneuron electrical properties (CHAPTER IV)?

3) Do motoneuron electrical properties recover to control values

upon reinnervation of the original muscle (CHAPTER III)?

4) What is the time course and pattern of recovery during

self-reinnervation (CHAPTER IV)?

5) Given a 'choice' of end organs, is there selectivity of

reinnervation on the basis of motor unit type (CHAPTER VI)?






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6) What are the limits of motoneuron influence upon muscle

properties (CHAPTER VI)?

7) Does the expression of motoneuron electrical properties depend

upon the particular muscle innervated; i.e. is there a regulatory

influence of muscle, upon motoneurons (CHAPTER VII)?
















CHAPTER II
METHODS



Experiments were performed on 51 adult cats (22 normal cats, 29

experimental). Animals were anesthetized to a depth sufficient to

maintain areflexia, during all surgical and experimental procedures. All

animals were adult at the time of the initial surgery.

Initial Surgery

Animals were anesthetized with a gaseous mixture of oxygen, nitrous

oxide, and halothane. Under aseptic conditions, the MG and LO-S nerves

of the left hind leg were surgically isolated within the popliteal

fossa, sectioned about 15-25mm proximal to the triceps surae muscles,

and the proximal stumps of each nerve were resutured to the distal stump

leading to the muscles) not innervated by that nerve originally

(X-reinnervation; Fig. 2-1) or to the stump leading to the original

muscle (self-reinnervation). In some experiments this procedure was also

carried out on the right leg. To direct axonal growth, 15mm long sleeves

of Gore-Tex (30um internodal distance) were used (Young et al. 1984;

see their Fig. 2). The two nerve ends were joined and their orientation

fixed with two 9/0 sutures through the epineurium.

Following recovery animals were maintained in pairs in 90x90cm

cages. The animals received care and were exercised daily outside of the

cage.


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LG-S NERVE


MG NERVE


Figure 2-1. Schematic diagram of cross-reinnervation surgery. Location
of Gore-Tex sleeves shown as black cylinders.









Acute ExDeriments

The acute experiments utilized techniques and protocol published

previously (Fleshman et al. 1981; Zengel et al. 1985). Operated animals

were studied three weeks to 11 months following the initial surgery.

Animals were anesthetized via intraperitoneal injection of sodium

pentobarbitol (35mg/kg). Leg, body, and spinal cord temperatures were

maintained at 350-370C with a heat lamp and heating pad.

The unoperated, normal M3 population derives from 16 animals (15

females, one male; 2.0 to 6.4 kg, mean 3.5 kg). The normal LG and soleus

data derive from 15 animals (eight animals were the same as for M3; 14

females and one male; 1.9 to 3.8 kg, mean 2.8kg).

Four acute experiments were performed on animals (all females, 2.8

to 3.4 kg, mean 3.0 kg) whose H3 nerve was sectioned and had not yet

reinnervated muscle (no-re; 20-35 days post-operative). Eleven animals

were examined at various times after self-reinnervation of M3 muscle.

Two animals (both female, 3.0 and 3.3 kg) were investigated five to six

weeks following the initial surgery (low-re; these animals had whole

muscle twitch tensions of less than 200g-wt). Two additional animals

(both female, 2.9 and 3.3 kg) were examined nine to ten weeks

post-operative (med-re). These animals had whole muscle twitch tensions

approximately 50% of long self-reinnervated MG. Five animals (four

female, one male, 2.9 to 3.6 kg, mean 3.4 kg) were examined nine months

after self-anastomosis of the M3 nerve (long-re). A final two animals

(both female, 3.5 and 2.9 kg) were examined whose recovery was

intermediate between the low-re and med-re stages .







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Cross-anastomosis of the MG and LO-soleus nerves was accomplished

for 14 animals (all females). Four of these were investigated at

nine-to-ten weeks (Afme, 2.0 to 2.8 kg, mean 2.4 kg). The remaining ten

animals were examined nine-to-11 months post-operatively (longX, 2.2 to

3.7 kg, mean 2.9 kg).

Whole Muscle Twitch

Prior to single unit recording, whole muscle twitch contractions

were obtained by stimulating the MG or LG-S nerve, with the MG, LG, or

soleus tendon attached to a force transducer. Each muscle was tested in

response to each of the two nerves, against a passive tension of

100g-wt., and also in response to the nerve eliciting the greater

tension, at the length providing maximal tension. The muscles were

activated tetanically before each measurement to potentiate the twitch

response. In some animals whole muscle twitches were obtained again

following single unit studies. The MG and LG muscle were then completely

separated and the series of twitches recorded again.

Motor Unit Studies

MG motoneurons were identified by antidromic stimulation of the MS

nerve, central to the nerve sleeve (innervates MG in

self-reinnervation, LG and soleus in cross-reinnervation). Motoneurons

were impaled with 3M KCl-filled microelectrodes, with impedances in the

4-12 Mohm range (measured at 1KHz). The muscle innervated by the MG

motoneuron, was differentiated by intracellular current injection, and

locating the muscle response.

Methods for obtaining and analyzing motoneuron and muscle unit data

were as reported previously (Fleshman et al. 1981; Zengel et al. 1985).






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We recorded antidromic action potential conduction time, rheobase, input

resistance [by the direct method of measuring voltage deflection by

means of a bridge circuit while injecting a InA current, without

correction for nonlinearities, (Ito and Oshima, 1965)], and

after-hyperpolarization potential (AHP) for each cell. The derived value

rheobase/input resistance was calculated for each motor unit because

that value (in conjunction with AHP) has been shown to precisely

categorize the unit type to which a motoneuron belongs in normal M3

(Zengel et al. 1985). The time for the AHP to decay to half-maximum

amplitude (AHP half-decay time) was measured. Only motoneurons with

action potential amplitude of 60mV, or greater, were used for analysis

of motoneuron electrical properties. Muscle unit contractile data were

obtained from a few units where motoneuron action potentials were less

than 60mV.

The contractile responses of motor units were activated by

intracellular current injection at the motoneuron, and measured using

the methods of Burke et al. (1973). Muscle unit contractile responses

were measured against a passive tension of 100g-wt. The following

responses were recorded: unpotentiated twitch; potentiated twitch

(following 100Hz tetanic stimulation); unfused tetanus ('sag'); tetanic

tension (600 ms at 100 Hz for fast units, 1500 ms at 100Hz for slow

units) and the fatigue test (Burke et al. 1973).

Motor units were classified on the basis of potentiated twitch

time-to-peak (fast units had a time-to-peak of potentiated twitch of

40ms or less and exhibited sag, slow units had time-to-peak of > 40ns

and did not sag) and fatiguability (FF units had fatigue index of (






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0.25, S and FR units had fatigue indices of > 0.75, and FI units were in

between) as type FF, FI, FR, or S (Burke et al. 1973; Fleshman et al.

1981). Units with twitch time-to-peak in the range 35-45ms were

classified as fast or slow by the presence (fast) or absence (slow) of

"sag" (Burke et al 1973) in an unfused tetanus (inter-stimulus interval

= 1.25X time-to-peak).

Muscle Histochemistrv

Animals were sacrificed by overdose of sodium pentobarbitol. The

left MG, LG, and soleus muscles were excised, cleaned of excess

connective tissue, blotted dry, and weighed to the nearest 0.1g. For MG

and soleus, a 10mm block of tissue was removed from the thickest part of

the muscle belly, cutting perpendicular to muscle fiber direction.

Lateral gastrocnemius muscles were cut into three blocks: proximal,

middle, and distal, each including approximately one third of the muscle

length. The blocks of muscle were fixed to a piece of cork with gum

tragacanth. Muscle orientation was carefully noted. The cork and muscle

were then immersed in isopentane cooled to -1600C by immersion in liquid

nitrogen. The tissue was then placed in a cryostat maintained at -200C.

After a few minutes drying time, the frozen tissue was wrapped in

parafilm, placed in a plastic vial, and stored until cut and stained.

Muscle histochemistry was performed within one week of the

acute experiment. The tissue and cork were mounted on a cryostat chuck

with OCT compound. Ten um thick serial sections were cut from each

block, and stained for myosin ATPase (preincubated at pH 10.3 and 4.2,;

Padykula and Herman, 1955; Guth and Samaha, 1970), Nicotinamide adenine






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dinuoleotide diaphorase (NADH-D; Novikoff et ll. 1961) and

A-glycerophosphate dehydrogenase (OPD; Wattenberg and Leong, 1960).

Muscle fiber types were classified using the system of Peter et al.

(1972). This scheme designates fibers as fast or slow, on the basis of

alkaline myosin ATPase (dark= fast; light = slow). Fast fibers are

further subdivided into fast glyoolytic fibers (FG), staining light for

NADH-D and dark for GPD, and fast oxidative glycolytic (FOG), staining

dark for both NADH-D and GPD. Slow (SO) fibers stain dark for NADH-D and

light for GPD.

The distribution of muscle fiber types was determined from

projections of the original slides with a microprojector. For LG,

approximately 600-1000 fibers were analyzed for myosin ATPase and

NADH-D, from each of the four compartments of the muscle (English and

Letbetter, 1982a,b). The LGm compartment was analyzed in the most

medial portion of the proximal section (separated by tendon). The LG2

compartment was located just lateral to the LGm compartment, in the

middle section. LG1 was analyzed from the distal section, near the

lateral border (lateral to tendonous inscription). Finally, LG3 was

located near the medial border of the distal section (see English and

Letbetter, 1982b; their Fig. 3). For soleus, the number of fibers with

"fast" (type II; Engel, 1970) staining characteristics were counted. For

MG approximately 1000 fibers from each of three separate areas (single

cross-section) were sampled. Muscle fiber areas were determined by

planimetry from photographs for 25-50 fibers of each type (FG,FOG,SO)

for each compartment of LG and for each MG or soleus muscle.






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Following single unit studies in two long self-reinnervation

experiments in which both M3 nerves had been sectioned and

re-anastomosed, a single type FF motor unit was isolated on the

contralateral side of the animal by intracellular impalement. After

measurement of motor unit properties, the unit was repetitively

activated using the same stimulus regime as for the fatigue test. This

was repeated over approximately one half hour, with one minute rest

between each two minute stimulation period, in order to deplete the

muscle fibers of that motor unit of their stores of glycogen (Edstrom

and Kugleberg, 1968; Kugleberget al. 1970). These muscles were prepared

as above for muscle histochemistry. An additional serial section was

stained for glycogen (PAS stain). The muscle was sectioned until the

section with the maximum number of depleted fibers was located. Serial

sections for myosin ATPase and NADH-D were examined to determine whether

reinnervated motor units were homogeneous with respect to muscle fiber

types and to examine their distribution.

Statistical Considerations

All statistical analysis was performed with programs written in the

language of the Statistical Analysis System (SAS; Helwig and Council,

1979). Mann-Whitney U-test was used to determine differences between

two means. Wilcoxon's multiple range test was used to compare multiple

means. Post-hoc analysis with Tukey's range test was used to determine

which specific means were different. Significant differences between the

proportions of motor unit or muscle fiber types were determined by the

chi square test, and comparison to the hypergeometric distribution. An

alpha value of 0.01 was considered to be significant as different






-14-


oontrol samples ooassionally differed at the 0.05 level. SAS programs

were executed with the facilities of the Northeast Regional Date Center

at the University of Florida.















CHAPTER 3
NORMAL MG AND LONG-TERM SELF-REINNERVATION OF MG


Introduction



Since the initial cross-reinnervation study of Buller et al. (1960),

attention has been focused on the role of the motoneuron in dictating

muscle properties. Numerous studies of contractile properties at the

whole muscle (Close, 1965; Close and Hoh, 1969; Luff, 1975; Prewitt and

Salafsky, 1967) and single unit levels (Bagust et al. 1981; Burke, 1980;

Burke et al. 1979; Chan et al. 1982; Dum et al. 1979; Lewis et al. 1982)

have supported the original hypothesis of neural determination of muscle

properties, and extended its documentation to finer levels of

organization.

In addition, experiments in which muscles of mixed fiber type

composition were subjected to chronic electrical stimulation indicate

that the frequency and/or amount of muscle activity are important

factors in the determination of muscle contractile properties (Eerbeek

et al. 1984; Goldring et al. 1981; Hudlicka et al. 1982; Lomo et al.

1974, 1980; Salmons and Vrbova, 1969; Smith, 1978; see additional

references in Pette, 1984; Salmons and Henrikson, 1981).

All of these studies (cross-reinnervation and stimulation) indicate

that it is easier to change a fast muscle to slow than the reverse. The

chronic stimulation experiments suggest that neural activity plays a

-15-






-16-


major role in determining muscle fiber characteristics, but there appear

to be limits to the extent of this influence.

Only two laboratories have previously related motor unit properties

to motor unit type following reinnervation. The motoneuron properties

investigated were axonal conduction velocity (Burke, 1980; Gordon and

Stein, 1982a,b), AHP duration (Burke, 1980), and extracellular action

potential amplitude (Gordon and Stein, 1982a,b). All studies reported

recovery to control levels by nine months to one year post-operative.

Kuno et al. (1974b) reported that five months following the

initial surgery, conduction velocity, resting membrane potential, ARP

duration, and action potential overshoot were incompletely recovered in

reinnervated cat M3 and soleus motoneurons. In that study all MH

motoneurons were considered 'fast' and soleus motoneurons were

considered 'slow. Czeh et al. (1978) suggested that AHP duration is

regulated by retrograde trophic messages from muscle. There is some

indication of a muscle influence on axonal conduction velocity as well

(Burke, 1980; Lewis et al. 1978). Thus, in addition to neural induction

of muscle properties there may also be retrograde influence upon the

motoneuron from the muscle.

In light of the differences in normal animals between motor unit

types in their motoneuron electrical and muscle unit contractile

properties (c.f. Fleshman et al. 1981; Zengel et al. 1985), it is of

interest whether motoneuron electrical properties, muscle unit

contractile properties, and their interrelationships, are restored

following self-reinnervation. The difference between slow soleus and

the overall M3 motoneuron sample in the studies of Kuno and colleagues






-17-


(Kuno, 1984) suggests that fast and slow motor units in a mixed muscle

may show different responses to axotomy and subsequent reinnervation.

A second question in reinnervation of adult skeletal muscle is

whether all motoneurons have an equal ability to reinnervate and

maintain connections with muscle fibers. Lewis et al. (1982) observed a

dramatically increased amount of type I (associated with slow motor

units) muscle fibers in one cat flexor halluois longus (FHL) muscle

three years following self-reinnervation. In combination with the

earlier observation of a reversal in the normal relationship of axonal

conduction velocity to motor unit tension production in soleus muscle

cross-reinnervated by the FDL nerve (slower units larger than fast

units; the reverse of normal FDL; Bagust et al. 1981), this suggested

to these workers that slow axons might have a competitive advantage

over fast axons in reinnervating adult slow muscle fibers.

We here use an intracellular approach to the model of

self-reinnervation of the M1 muscle in the cat, to explore the limits

of regulatory interactions between alpha-motoneurons and the muscle

fibers they innervate. Specifically, three questions are addressed.

First, are motoneuron and muscle properties reestablished (overall and

within motor unit types) following nine months self-reinnervation?

Second, is there evidence for a competitive advantage of any motor unit

type in reestablishing motor unit size or numbers? Finally, to what

extent are muscle unit properties dictated by the innervating

motoneurons and vice ersa; that is, are the normal relationships

between motoneuron and muscle properties restored?






-18-


Results

Effects of Cage Time

Since some of the animals in this study were caged for nine-months

prior to the acute experiments, it was important to control for any

effects of cage-time per se. Data were compared from non-caged animals

(11 cats, all female, 2.0 to 6.4 kg, mean weight 3.7 kg) and cats caged

from 151 to 284 days (5 oats, four females and one male, 2.1 to 3.6 kg,

mean weight 2.9 kg). There were no differences in the distribution of

motor unit types between non-caged and long-caged cats.

The only differences in motoneuron properties (p<0.01, Mann-Whitney

U-test) between non-cagcd and long-caged cats were rheobase and

rheobase/input resistance of the population as a whole (across motor

unit types), and input resistance in type S motor units (Table 3-2).

Similarly, contractile parameters did not differ between non-caged and

long-caged cats at the 0.01 alpha level (Table 3-3). We conclude that

the effects of cage time were minimal and the non-caged and long-cage

groups will be considered together as the normal control group for the

remainder of this paper.

Normal Data

Table 3-1 lists values for muscle wet weights and whole muscle

twitches for normal and self-reinnervated MG muscles. Normal MG values

for muscle weight, muscle weight/body weight, and speed and

tension-related parameters were similar to those obtained for cat MG in

other studies (Gardiner et al. 1978; Mayer et al. 1984; Sacks and Roy,

1982, Spector et al. 1980).






-19-


Table 3-1. Whole Muscle Properties: NORMAL and Nine Month
Self-reinnervated. a,c

NORMAL LONG-RE


Twitch
Twitch
Twitch
Twitch
Muscle
Muscle
Twitch


Time-To-Peak b ms)
Half-Rise Time b(ms)
Half-Relaxation Time (ms)
Tension b(g-wt.)
Weight (g)
Wt./Cat Wt. (g/kg)
Tension/Cat Weight (g-wt./kg)


32.11
110
222
2123151
9.01
3.00
74265


333
100
252
16091276
7.01'
2.30*
488.66*


a. MeansSE (for NORMAL n=14;
b. Twitch at muscle length at
c. = significant difference


for LONG-RE n=4).
which maximum tension was obtained.
from control (p<0.05);






-20-


The normal population (non-caged plus caged, see Methods) consisted

of 48% type FF units, 4% type FI, 24% type FR, and 2i% type S units

(Table 3-6). This is similar to that reported by Burke et al. (1973),

and Fleshman et al. (1981).

Normal MG muscle contained 56% type FG, 23% type FOG, and 21% type

SO fibers (Table 3-6; see also Ariano et al. 1973; Burke and Tsairis,

1973). There were no differences between non-caged and long-caged cats

with respect to muscle fiber type distribution.

In cat MG, type FF muscle units are comprised of type FG muscle

fibers (or IIb), type FR units of type FOG fibers (IIa), and type S

units of type SO fibers (I; Burke and Tsairis, 1973). Table 3-6

estimates relative innervation ratios for each motor unit type.

In general, normal values for motoneuron electrical properties

(Table 3-3) and muscle unit contractile properties (Table 3-2) were

similar to those reported previously (Burke et al. 1973; Fleshman et al.

1981; Zengel et al. 1985).

Control vs. Reinnervated

Acute physiological experiments were performed on four animals

(three female, one male; 2.9 to 3.6 kg, mean 3.4 kg) nine months after

the initial surgery. An additional animal (female, 2.7 kg) was used for

muscle histochemistry only. The normal distribution of motor unit types

was reestablished, confirming results obtained by Burke and colleagues

(Burke, 1980; Burke I 3aL 1979; Dum et al. 1979, in press b) in

self-reinnervated cat FDL, and by Gordon and Stein (1982a) in

self-reinnervated cat M3 (Table 3-6).






-21-


Whole Muscle

Following nine months recovery from the initial surgery, HO muscle

weights were about 70% of unoperated normal MD. Whole muscle twitch

time-to-peak as well as half-rise and half-relaxation times were normal

(Table 3-1; Bagust and Lewis, 1974; Buller et al. 1960; Burke, 1980;

Burke et al. 1979; Chan et al. 1982; Dum et al. 1979, 1985b; Gordon and

Stein, 1982b). In the operated animals muscle weight was significantly

reduced as was muscle weight/body weight. Twitch tension was also lower

in reinnervated animals (p<0.05), but tension /muscle weight was

unchanged (Table 3-1). These results are similar to those of Chan et al.

(1982), Bagust et al. (1981), and Lewis et al. (1982), but Gordon and

Stein (1982b) and Burke (1980) saw complete recovery of muscle weight

and twitch tension.

Muscle Unit Contractile Properties.

There were several differences in contractile properties between

normal and self-reinnervated animals (Table 3-2). Overall, across motor

unit types, mean maximum tetanus was lower in the reinnervated group (23

vs. 35g-wt.; Table 3-2). This trend was still present when maximum

tetanus was normalized by body weight, although no longer statistically

significant (p>0.05). This overall effect reflects the significantly

lower maximum tetanic contractions of type FF units (also significant

for normalized values). Types FR and S unit tensions were normal.

Consistent with the mean values, there were few units with large maximum

tetanic contractions in the reinnervated muscles (Fig. 3-10). We saw no






-22-


Table 3-2. Muscle Unit Contractile Properties. (Long-caged vs. Non-caged;
Normal vs. Nine month self-reinnervated). abc
FF Fl FR S ALL

TWITCH TIME-TO-PEAK (ms) d


24_+1
271
26+1
271


(10)
(20)
(30)
(17)


505 (4)
6014 (10)
584 (22)
55+3 (18)


301 (35)
362 (80)
342 (115)
34-2 (70)


TWITCH TENSION (g-wt.)


1+0
10
1+0
1+0
1.t0


(10)
(10)
(28)
(17)


0.20
0.30
0.2+0
1.0+0


(4)
(18)
(22)
(18)


90 (35)
8.1 (78)
91 (113)
51 (70)'


H CTIWT HALF-RELAXAT N


23+2
271
26+1
252


(9)
(20)
(29)
(16)


5711 (3)
749 (16)
748 (19)
51+4 (15)


262 (33)
363 (78)6
332 (111)
32r2 (66)


MAXIMUM TETANIC TENSION (g-wt.)


161
13+4
131
13+3


(25)
(20)
(45)
(17)


9t2
31
71
61


(22)
(17)
(39)
(16)


383 (96)
313 (78)
352 (175)
23+2 (67)'


FATIGUE INDEX


1.10
1.00
1.0+0
1.2+0


(25)
(20)
(45)
(17)


1.0+0

1.1+0
1.1+0


(21)
(17)
(40)
(13)


0.60 (94)
0.50 (79)
0.60 (174)
0.60 (63)


a. = Significant difference from NON-CAGE at 0.05 level; *" 0.1 level
b. + = Significant difference from NORMAL at 0.05 level: ++ 0.01 level
c. Means SE (number of units).
d. Potentiated Twitch.
e. NORMAL = NON-CAGE + LONG-CAGE.


NON-CAGE
LONG-CAGE
NORMAL MG
LONG-RE


29.1
291
291
28.1


(17)
(39)
(56)
(32)


292
263
28+2
255


NON-CAGE
LONG-CAGE
NORMAL MG
LONG-RE


192
171
181
101


(17)
(39)
(56)
(32)'


4.2
42
411+
26


NON-CAGE
LONG-CAGE
NORMAL MG
LONG-RE


232
251
24.1
28.t2


(17)
(39)
(56)
(32)


30-3
231
272
24+5


NON-CAGE
LONG-CAGE
NORMAL MH
LONG-RE


664
553
613
374


(45)
(39)
(84)
(31)'


22+1
188
203
20.0


NON-CAGE
LONG-CAGE
NORMAL MG
LONG-RE


.040
.014+0
0.030
0.1+0


(44)
(38)
(82)
(30)*


.590
.340
0.50
0.5+0


TWTTCH BALF- RE7-AXATTON-~


TIME (ms) d






-23-


evidence of greatly enlarged motor units with reinnervation (Chan et al.

1982; Gordon and Stein, 1982a,b; but see Rosenfalck and Buchthal, 1970;

Yahr et al. 1950). Most studies of reinnervated muscle have reported

decreased mean tension with increased variance [Bagust and Lewis, 1974;

Burke, 1980 (oat flexor digitorum longus: FDL); Chan et al. 1982 (oat

flexor hallucis longus: FHL)]. Distributions of twitch and tetanic

tension in this study were similar to those of normals in range for all

types except type FF where distributions were shifted to lower values

(Fig. 3-1A,C).

Mean potentiated twitch amplitude was smaller in self-reinnervated

MH, but not significantly so (Table 3-2). The overall distribution was

similar to that of normals, except there were few large twitch units

within type FF (Fig. 3-1A). Gordon and Stein (1982b) reported recovery

of mean twitch tension, with reinnervated units showing higher variance

than normals. Bagust and Lewis (1974), Bagust et al. (1981), and Lewis

et al. (1982) found little change in mean twitch tension, although the

distribution included a few very large units and many very small units.

Part of the difference between their results and ours may be due to

their expression of motor unit tension relative to whole muscle tension.

Absolute motor unit sizes were not abnormally large compared to normal

values.

In general, type FF units produced less tension and type S units

more tension than normals (Table 3-2; also true if normalized by cat

weight). The increased type S unit twitch amplitude, without increased

tetanus resulted in significantly increased twitch/tetanus ratio. We saw

no difference in twitch/tetanus ratio between normal and reinnervated

M3.


























r
FF




is













Is
5t. --.._ .







I






C T iL
I n s



a- T ** -DEER TI ME (s 0


a
Ir
IS



I .1 1.1 I.I I2. 1 3.
INPUT RESISTANCE lMobsh


D





FR

IS


o I .N it



RXONAL COOLCTICN VELOCTT WI/s)


Figure 3-1. Frequency histograms for normal (unfilled) and nine
month self-reinnervated (filled) MG motoneuron properties.
(A) Rheobase (B) Input resistance (C) AHP half-decay time (D)
axonal conduction velocity. See text for explanation.


-24-






-25-


There were no differences between reinnervated and normal muscle

units in twitch time-to-peak (Bagust and Lewis, 1974b; Burke, 1980; Chan

et al. 1982; Gordon and Stein, 1982a) or half-relaxation time (Bagust

and Lewis, 1974b). There were no differences in the frequency

distributions for the reinnervated units' twitch time-to-peak or fatigue

index (Fig. 3-1B,D) compared to normals. There was a significant

increase in mean fatigue index in type FF units of reinnervated animals

(0.06 vs. 0.03), confirming a general impression of a "residual" tension

following the fatigue test in these animals (unpublished observations).

Reinnervated motor units were similar to normal units in the pattern

of differences between motor unit types for contractile properties

(Table 3-4). The exceptions were that reinnervated types FR and S unit

fatigue indices did not differ, and the only difference for

twitch/tetanus was between types S and FR units.

In reinnervated units the overall correlation between twitch

time-to-peak and tetanic tension was -0.47 (p< 0.0001), similar to

normals (-.40, p< 0.0001), and confirming Bagust and Lewis (1974). The

correlation was -0.53 within reinnervated type S units (p< 0.02; not

significant in normals). Types FF and FR units show no correlation

between these properties in normals or reinnervated animals.

The overall correlation between twitch time-to-peak and twitch

amplitude was weak (-0.32, p<0.004) for self-reinnervated units (vs.

-.29, p< 0.003 in normals) and nonsignificant within any motor unit type

in either population. Gordon and Stein (1982a) reported a restoration

of the normal negative correlation between twitch time-to-peak and

twitch amplitude with reinnervation.






-26-


Speed- and fatigue-related properties were normal in mean values and

distribution nine months' following section and re-suture of the MH

nerve. The relationships between time-to-peak and tension were also

normal. The exception to complete restoration of normal properties was

that type FF units did not fully recover ability to generate tension.

Motoneuron Electrical Properties

There were no significant differences in motoneuron properties

between operated animals and normals (p<0.01). At the 0.05 alpha level

only rheobase in FF units, and rheobase/input resistance in S units

reached significance (Table 3-3). Significant differences between motor

unit types in reinnervated motor units were similar to normals (Table

3-4). Exceptions include input resistance, where FR units were not

significantly different from FF or S units, and rheobase, where FR and S

units differ at p<0.05 only.

There was no significant difference in axonal conduction velocity

between regenerated and normal MG motoneurons (Table 3-3). This was also

seen by Gordon and Stein (1982a) for MH self-reinnervated for 9 months.

Kuno et al. (1974b) reported that after 4 months, regenerated MG

motoneurons' axonal conduction velocity had not quite recovered to

non-caged levels. This difference of results may be due to the shorter

recovery time in the Kuno et al. (1974b) study. Consistent with this,

Lewiset al. (1978) reported lower than non-caged conduction velocity in

self-reinnervated FDL axons at 6 months but not at two years following

the initial surgery.






-27-


Table 3-3. Motoneuron Electrical Properties. (Long-caged vs. Non-caged; Normal
vs. Nine month self-reinnervated). a, ,8


RHEOBASE (nA)

NON-CAGE
LONG-CAGE
NORMAL MG d
LONG-RE


221
18+1
201 1
2041
24t?


(40)
(20)
(70)
(33)


18t2
14-+1
16+11
18+3


S1+tl
101
11+1
111


(23)
(14)
(37)
(18)


(20)
(14)
(34)
(19)


161 (86)
131 (61)"
14+1 (147)+
151 (73)+


INPUT RESISTANCE (Mohms)


NON-CAGE
LONG-CAGE
NORMAL MG
LONG-RE


0.60
0.70
0.60
0.7+0


(33)
(23)
(56)
(22)


0.60
1.4
0.90
0. 70


1.00
1.10
1.1+1
1.2+0


(17)
(13)
(30)
(10)


1.20
1.90
1.50
1.30


(15) 0.90 (67)
(13)** 1.20 (50)
(28) 1.0+0 (117)+
(11) 1.00 (45)+


RHEOBASE/INPUT RESISTANCE (nA/Mohms)


NON-CAGE
LONG-CAGE
NORMAL MG d
LONG-RE


382
292
352
401+4


(32)
(23)
(55)
(22)


28+1
14
235
3312


152
101
121
10+2


(17)
(13)
(30)
(10)


5+1
ail
1+1
461
61


(15)
(13)
(28)
(11)


232 (66)
16+2 (50)
212 (117)+
233 (45)+


AHP HALF-DECAY TIME (ms)


NON-CAGE
LONG-CAGE
NORMAL MG d
LONG-RE


212
231
22+1
20+1


(25)
(29)
(54)
(33)


150
221
192
23+1


241
24+1
251
242


(17)
(14)
(31)
(18)


431
514
493
443


(14)
(14)
(28)
(19)


261 (48)
30,2 (60)
28.1 (118)
271 (73)


AXONAL CONDUCTION VELOCITY (ms)


NON-CAGE
LONG-CAGE
NORMAL MG d
LONG-RE


99.2
961
971
96+2


(16)
(39)
(55)
(33)


1042
958
1004
945


1032
992
992
96-2


(7)
(18)
(25)
(18)


854
802
812
84+2


(4)
(20)
(24)
(19)


981 (31)
921 (80)
941 (111)+
901 (73)+


* = Significance at 0.05 level from NON-CAGE; ## 0.01 level.
+ = Significance at 0.05 level from NORMAL; ++ 0.01 level.
Means SE (number of units).
NORMAL MG = NON-CAGE + LONG-CAGE.






-28-


Table 3-4. Results of Tukey's Studentized Range Test: Significance of
Differences Between Motor Unit Types: NORMAL VS. Nine Month
Self-reinnervated MG (LONG-RE).




NORMAL LONG-RE


Axonal Conduction Velocity
Rheobase

Input Resistance

Rheobase/Input Resistance

AHP Half-Decay Time
Twitch Amplitude a,b
Twitch Time-To-Peak b
Twitch HRT b,c
Maximum Tetanic Tension a
Fatigue Index
Twitch/Tetanus d


a) The same result was obtained
weight.
b) Potentiated twitch.
c) HRT = half-relaxation time.


(F,R)>S 0.01
F>R>S 0.01

F

F>(R,S)
F>R>S
(F,R) F>(R,S)
(F,R) (F,R) F>(R,S)
F F>(R,S)


0.01
0.05
0.01
0.01
0.01
0.01
0.01
0.01
0.01


(F,R)>S>N
F>(R,S,N)
F>R>(S,N)
F<(S,N)
F F>(R,S,N)
F>(R,S,N)
(F,R,N) F>(R,S)
(F,R) (F,R) F>(R,S)
F<(R,S)
(F,S)>R


with raw data and data normalized for body


d) Twitch/tetanus = unpotentiated twitch/maximum tetanus.


0.01
0.01
0.05
0.01
0.05
0.01
0.05
0.01
0.01
0.01
0.01
0.01
0.01
0.01






-29-


All motor unit types had ranges of AHP half-decay times similar to

normal (Fig. 3-2). Kuno et al. (1974b) found that regenerated Mi

motoneurons had AHP durations that were not significantly longer than

normal (82 vs 75ms). In contrast, regenerated soleus motoneurons

(virtually all slow in normals) had AHP durations that were

significantly shorter than normal (123 vs 150ms). Thus the type S

motoneurons of the mixed muscle MG appear to behave differently in this

respect than soleus type S motor units following regeneration.

Kuno et al. (1974b) found that 45-60% of five-month regenerated MG

motoneurons had values for AHP duration/axonal conduction velocity

within the normal range. We found that 96% (44/46) of regenerated MG

motoneurons were in the normal range with respect to AHP half-decay time

and axonal conduction velocity (Fig. 3-3) and there was little overlap

between the distribution of fast and slow units (35/36= 97% of

regenerated fast motoneurons in the normal fast range; 13/18 = 72% of

regenerated slow motoneurons in the normal slow range). Axonal

conduction velocity decreased as AHP half-decay time increased in

regenerated motoneurons (r= -0.42, p<0.0001; vs. -0.52, p<0.0001 in

normals). Within reinnervated type S units the correlation was -0.69

(p<0.0001; -0.51, p<0.03 in normals), and there was no relationship for

types FF and FR units as in normals.

The distribution of values for rheobase was indistinguishable from

that of normals (Fig. 3-2A). The same was true for input resistance,

with the possible exception of a lack of large input resistance (>1.5

Mohm) cells in S units of the operated animals (Fig. 3-2B). This may

represent an artifact of sampling.































25 FR
I5

Is

Is M as s ?S
TWITCH TIME-TO-PEAK (as)


,.n



is n

S L


TETANIC TENSION tg-wtl


B -r


35
1 5
ih- n. ,






TWITCH TENSION tg-wtl
g Is






inL -.. ..
IS S





v a 1 A-Ns

FATIGUE INDEXX (TII 7 E S
TWITCH TENSION (g-vt)

D "
2 r










FRTISUE :cE3x (TICS IlU)


Figure 3-2. Frequency histograms for normal (unfilled) and nine
month self-reinnervated (filled) M3 muscle unit contractile
properties. (A) potentiated twitch amplitude (B) potentiated
twitch time-to-peak (C) tetanic tension (D) fatigue index. See
text for explanation.


-30-


A








iC





C







-31-


Figure 3-4A shows the relationship between rheobase and input

resistance in regenerated motoneurons (3-4B) as compared to normals

(3-4A). Figure 3-4B illustrates that, as in normals, motoneurons

segregated according to type on the basis of rheobase: input resistance

ratio. The correlation between log rheobase and input resistance was

-0.63 (normal, -0.60, p<0.0001; see also Fleshman et al. 1981; Zengel

et al. 1985). No significant correlations were found within any motor

unit type in normal or reinnervated units.

The criteria used by Zengel et al. (1985) were applied to these data

to estimate motoneuron type (AHP half-decay time < 30ms = fast, AHP

half-decay time > 30ms = slow; rheobase/input resistance <7 = S,

rheobase/input resistance > 18 = FF, with FR motoneurons having

rheobase/input resistance between 7 and 18). Based on these criteria

there was an 86% agreement in normal MG, between motoneuron type and

motor unit type (determined by contractile properties). When these same

criteria were applied to the reinnervated motor units, there was an 841

agreement between motor unit type contractilee) and motoneuron type

(above criteria).

Thus, after nine months, regenerated MG motoneurons had normal mean

values and frequency distributions for membrane electrical properties.

Relationships between motoneuron electrical properties were normal, and

motoneuron type (defined by electrical properties) accurately predicted

motor unit type (defined by contractile properties).







-32-


'U


XON CO CION VELOCITY
XONIL CONOJICTION VELOCITY (/.s)


I I I


U S
n






I I I I I

RXONL COXOJCTION VELOCITY (./s)


Figure 3-3. Relationships between AHP half-decay time and axonal
conduction velocity in normal and nine month self-reinnervated MG
motoneurons. (A) Normal M3. Note overlapping but largely separate
distributions for slow (S) and fast (F=FF, R=FR, I=FI) motoneurons.
Solid lines outline the distribution of slow (upper) and fast
motoneurons. (B) Nine months self-reinnervated MR. Note precise
reestablishment of distributions for slow (S) and fast (F=FF, R=FR,
I=FI) motoneurons. Most non-contracts (N) fall outside the normal range.
Solid lines outline the distribution of slow (upper) and fast (lower)
motoneurons in controls.


4 -


I II I






-33-


Non-Contracts

Unlike those in the normal population, there were a few motoneurons

(8/81) in the self-reinnervated animals which, when stimulated, did not

elicit measurable muscle contractions. We have termed these cells

"non-contracts" as we cannot rule out their being .-fusimotor in nature

(Gregory et al. 1982). It is unlikely that these are gamma motoneurons

as they received monosynaptic input from the LG-S nerve (Table 3-5). A

subpopulation within the regenerated motoneurons which did not elicit

muscle contraction has been observed previously (Bagust and Lewis, 1974;

Gordon and Stein, 1982b; Kuno et al, 1974b; Dum et al. in press).

Mean values and raw data for individual non-contracts' electrical

properties are seen in Table 3-5. In axotomized motoneurons, mean axonal

conduction velocity and the mean and range for rheobase decreased, while

mean input resistance was increased (Chapter 4; Gustafsson, 1974;

Gustafsson and Pinter, 1984; Kuno et al. 1974a). Mean AHP half-decay

time is little changed with axotomy, but the range was compressed from

both the high and low ends (Chapter IV; Gustafsson, 1974; Gustafsson and

Pinter, 1984; Kuno et al. 1974a). Some of the non-contracts at nine

months' self-reinnervation fit this pattern (cells 6,7,and 8) while

others show various combinations of properties within and outside the

range for normal motoneurons. Six of the eight non-contracts fell

outside the normal range of values for the ratio AFP half-decay time:

axonal







-34-


41r


lIE


I I I I


S


I.S I 1. 2 2.1 S
INPUT REISTFCE (Moh*sJ


1.1 4


M


I '
- pP
I,


*


ph

16I


**S I
I
a s


gI I l I I
I8 .5 1.I 2 2.1 I 1. 4
INPLT RESISTANCE IMoh.%)


Figure 3-4. Relationships between rheobase and input resistance in
normal and nine month self-reinnervated MG motoneurons. F= type FF
units, I=type FI, R=type FR, S=type S units, N=non-contract. (A) Normal
M3. NoLE segregation by motor unit type. (B) Nine months
self-reinnervated MG. Segregation by motor unit type is re-established.
The lack of high input resistance type S units may reflect sampling
variability.


Io


I ~~ I






-35-


conduction velocity (Fig. 3-4). It.is possible that some of the cells

with relatively normal properties were injured in dissection or have

made contact with intrafusal muscle fibers (Gregory et al. 1982).

Relationships Between Motoneuron and Muscle Unit Properties

In normal animals, the strongest correlation between a motoneuron

electrical property and muscle unit contractile property was between AHP

half-decay time and twitch time-to-peak (r= 0.74, p<0.0001 overall; 73).

This relationship was also present in reinnervated units (0.42,

p<0.0001). We saw no significant relationships within motor unit types

in operated or normal animals, in contrast to Zengel etal. (1985), who

found a significant correlation within S units of MG. Huiszar et al,

(1977) also reported a significant correlation between AHP duration and

twitch time-to-peak for soleus motor units.

In operated animals, as in normals, the correlations between axonal

conduction velocity and twitch (r = 0.23, p<0.04) or tetanic tension

(0.37, p<0.001), normalized by cat weight and expressed as log tension,

were weak ( 0.41, p<0.0001 for twitch; 0.44, p<0.0001 for tetanus;

2,29). There were no significant correlations between these variables

within any motor unit type in reinnervated units. In normals the

relationships were significant within type S units only (0.58 for

twitch, ns for tetanus).

The correlation between axonal conduction velocity and twitch

time-to-peak was -0.50 overall (p<0.0001) and -0.53 for type S units

(p<0.02). This compares to -0.52 and ns in normals. Gordon and Stein

(1982a) also showed restoration of the normal negative correlation



















Table 3-5. Data
Model.)


CELL


RHEOa


for Non-Contracts (Nine Month Self-Reinnervation


RNb RHEO/RNc HALFTIMEd


X
x
x
X
1.2
1.0
1.3
1.5
2.7


X
x
x
X
X
4.2
12.0
2.3
3.3
1.1


C.V.e

76
89
73
87
53
40
32
63


PSPf

1.4
x
X
1.0
1.6
1.0
1.5
2.3
0.4


MEAN VALUES +SE (NUMBER OF UNITS)


NON-CONTRACTS


REGENERATED


RHEOBASE (nA)
RN b (Mohm)
RHEO/RN c (nA/Mohm)
HALFTIME (ms)
AXONAL C.V. e(m/s)


71
20
5_4
30+1
64_7


151
10
233
271
901


(73)
(45)
(45)
(73)
(73)


RHEO = rheobase
RN = input resistance
RHEO/RN = rheobase/input resistance
HALFTIME = AHP half-decay time
C.V. = conduction velocity
PSP = composite monosynaptic Ia EPSP from LG-S nerve


-36-


141
10
212
28+1
941


(147)
(117)
(116)
(111)
(118)






-37-


between axonal conduction velocity and twitch contraction time in

self-reinnervated cat H3. In contrast, Bagust and Lewis (1974) reported

that the normal inverse relationship between time-to-peak and axonal

conduction velocity was lost in reinnervated muscles. While it is not

clear what can account for these differences, the present surgeries were

all performed on adult cats whereas Bagust and Lewis (1974) operated on

young cats of around one kg in weight.

In summary, the relationships between electrical properties of

self-regenerated motoneurons and contractile properties of

self-reinnervated muscle units were similar to those which exist between

normal motoneurons and muscle units.

Muscle Fiber Histochemistrv and Fiber Areas

Mean cross-sectional areas of nine months' self-reinnervated MS

muscle fibers were 3098um2 for type FG fibers (3873um2 normal), 2355um2

for type FOG (2264um2 normal), and 2296um2 for type SO fibers (1972um2

normal), with an overall average of 2738um2 (88% of normal = 3104um2;

Table 3-6). The areas of types FOG and SO fibers were similar to normal

values but type FG fiber areas were significantly below normal levels.

The reduced overall mean area was due to this lack of type FG recovery.

Burke (1980) found that after one year, the normal distribution of

muscle fiber types was present in FDL, although "type-grouping"

(Dubowitz, 1967; Romanul and Van Der Meulen, 1967) was evident (Burke,

1980). We found 54% type FG fibers, 15% type FOG fibers, and 31% type

SO fibers (56;23;21 in normals) and fiber "type-grouping" was evident

(Fig. 3-5). This overall distribution was significantly different from

normal (chi-square test, p<0.05). There was an increase in the






-38-


Table 3-6. Motor Unit Types, Muscle Fiber Types, and Innervation
Ratios (Normal and Nine Month Self-Reinnervated).

MUSCLE FIBER TYPES

Histoohemical Composition


FG FOG


SO n/N a


NORMAL
LONG-RE


Observed Mean Fiber Area (um2)


NORMAL
LONG-RE


Est. Number of Muscle Fibers.


68560 28160 25710
58320 16200 33480


EQUIVALENT MOTOR UNIT TYPE


% Of Population


NORMAL
LONG-RE

Est. Number in Pool

NORMAL
LONG-RE C

CALCULATED VALUES


Relative IR d

NORMA L
LONG-RE


FF+FI


48+4
46+4


134+11
122+11


FF+FI FR S


1.0
0.6


Number of Cells/Number of Animals.
(Physiological Cross-Sectional Area)/Mean Fiber Area.
Estimated as 280-[(280) (% Non-Contracts)].
Relative Innervation Ratio = % Motor Unit Type/% Muscle


Fiber Type.


3873
3098


4200/7
3000/5


ALL
3104
2738


NORMAL
LONG-RE


2264
2355


1972
2296


122400
108000


n/V a


176/16
72/4


ALL
280
265


0.9
1.2






-39-


proportion of type SO muscle fibers, and a decrease in fast muscle

fibers as a group (types FOO+FO). This alteration of proportions of

muscle fiber types was not as extensive as that reported by Lewis t

al. (1982), however, who presented histochemical data for one

self-reinnervated FHL, after over two years' recovery. In that muscle

80$ of the fibers were type I (equivalent to type SO) compared to the

normal FHL which is 86% type II (Ariano et al. 1973).

Physiological Cross-Section and Innervation Ratios

The physiological cross-section (CSA) of the whole MG muscle was

estimated as follows (ref. Dum et al. 1982):

CSA= (muscle weight)(cosA)

(muscle density)(muscle fiber length)

Muscle density was assumed to be 1.06g cm'3 (Mendez and Keys, 1960).

Values for A (angle of pennation) and muscle fiber length were assumed

to be 210 and 21mm, respectively, for both normal and reinnervated

muscles (Sacks and Roy, 1982). For the reinnervated muscle we estimate

physiological cross-section as 3.0cm2 (3.8cm2 in normals).

The number of muscle fibers in WM was estimated as physiological

cross-section/mean muscle fiber area (Burke and Tsairis, 1973). Mean

muscle fiber area was estimated as the sum of the area for each muscle

fiber type, multiplied by the frequency of that type in the population.

For normals, we estimated the number of muscle fibers in MS as 122,400.

This is lower than the 170,000 estimated by Burke and Tsairis (1973) for

cat M3, presumably they obtained higher values for A and fiber length,

as their value for mean fiber area was higher (3426um2 calculated from

Burke and Tsairis, 1973, vs. 3104um2 in this study).









The overall average innervation ratio (mean number of muscle fibers

innervated by an individual motoneuron; Eccles and Sherrington, 1930) is

calculated as the ratio of the number of muscle fibers in MG (estimated

as 122,400) to the number of MG motoneurons (280: Boyd and Davey, 1968;

Burke et al. 1977). In this study average innervation ratio for normal

MG was 436 (vs. 607: Burke and Tsairis, 1973). For reinnervated MG we

estimate average innervation ratio to be 429 (108,000/252; estimated

numbers of both muscle fibers and innervating axons were decreased;

Table 3-6). The relative innervation ratio for each type is the

frequency of a given muscle fiber type divided by the frequency of the

corresponding motor unit type (Dum et al. 1982; their eq.2).

Relative innervation ratios for reinnervated types FF+FI motor units

were 1.1 (vs. 1.1 normals), 0.6 for type FR units (vs. 1.0, although

Burke and Tsairis, (1973) reported 0.7 in normal MG), and 1.2 in type S

units [vs. 0.9 in normals; 0.9 in Burke and Tsairis, 1973). This may

reflect some competitive advantage of S units in capturing and/or

maintaining contacts with muscle fibers, although not to the extent

reported by Lewis et al. (1982).

Mean motor unit tetanic tensions (Table 3-2) recovered to normal

levels in type FR units despite reduced innervation ratios (see below)

suggesting increased specific tension (Table 3-6). Bagust et al. (1981)

reported altered muscle specific tension following reinnervation and

cite Hob (1974) as showing myofibril packing density was influenced by

innervation. Type S units recover to 67% of normal tension levels

despite increased numbers of fibers and unchanged area (suggesting lower

specific tension, Table 3-6). Type FF units recovered to only 64% of






-41-


normal tetanic tension (Table 3-2), primarily due to decreased muscle

fiber area (no change in innervation ratio).

Fig. 3-5 shows a single glycogen-depleted type FF motor unit from a

nine month self-reinnervated M3. Note the "clumped" nature of the

distribution and the homogeneity of muscle fiber type. There were 141

fibers counted in this unit, in the section with the maximal number of

depleted fibers. All were FG fibers, confirming previous studies (Burke,

1980; Gauthier et al. 1983; Kugelberg et al. 1970), in that muscle fiber

type was homogeneous within a single motor unit following long-term

reinnervation. Burke and Tsairis (1973) estimated that counting depleted

fibers from a single section of normal MG sampled 50%-75% of the whole

unit. From this relation we estimate that the unit in Fig. 3-5 contained

212-284 muscle fibers. This unit had a motoneuron rbeobase of 32, muscle

twitch time-to-peak of 30ms, 4.4 g-wt. twitch tension, 25 g-wt. tetanic

tension, exhibited 'sag', and had a fatigue index of 0.07. A second

glycogen depleted type FF unit had 365 fibers in the section with

maximal number (estimate of 548-730 muscle fibers). Rheobase was 16 and

twitch time-to-peak was 20ms. Tension data are unavailable. Again all

fibers were type FG. This unit also exhibited type-grouping, but not to

the extent of the unit in Fig. 3-5. The innervation ratios of these

depleted FF units fall within the range reported by Burke and Tsairis

(1973) for normal MG.

Since calculated innervation ratios were increased for type S motor

units (Table 3-6), type S motoneurons may be at advantage in innervating

and maintaining connections with muscle fibers following reinnervation

of a mixed muscle. Gycogen-depleted type FF units (n=2) fell within the
















AC


Figure 3-5. Photomicrographs of a single glycogen-depleted type FF motor
unit from a nine month self-reinnervated MG. (A) The entire unit (PAS
stain for glycogen, 70X) (B) PAS stain (100X) (C) myosin ATPase, pH 4.3
(100X) (D) NADH-D (10OX).






-43-


normal range for innervation ratios of unoperated normal type FF motor

units.

Discussion

In this study we tested whether motoneuron electrical properties,

muscle unit contractile properties, and the normal relationships between

them, would be restored nine months following section and re-anastomosis

of the MG muscle nerve in oats.

We have shown that the normal proportions of motor unit types are

re-established in self-reinnervated muscles (Burke, 1980; Gordon and

Stein, 1982a). Motoneuron electrical properties (rheobase, input

resistance, AHP half-decay time, and axonal conduction velocity), as

well as correspondence between motoneuron (electrical) type and motor

unit contractilee) type, are normal following nine months reinnervation.

As in controls, self-regenerated MG motoneurons segregate by motor unit

type with respect to the ratio rheobase:input resistance. This ratio, in

conjunction with AHP half-decay time, predicts motor unit types in

self-reinnervated as well as control MG (Zengel et al. 1985).

We found that axonal conduction velocity recovers to normal levels

following reinnervation (Burke, 1980; Burke et al. 1979; Gordon and

Stein, 1982a; Kuno et al. 1974b; Lewis et al. 1978). Kuno et al. (1974b)

showed that after five months self-reinnervation, action potential

overshoot, resting membrane potential, and conduction velocity were

normal or nearly normal in M5, while AHP duration had not recovered

completely. Our study confirms these earlier reports, and extends the

documentation of recovered motoneuron properties to motoneuron rheobase,

input resistance, and their relationship. Previous works have not






-W4-


distinguished between motor unit types with respect to motoneuron

properties [except axonal conduction velocity (Burke, 1980; Gordon and

Stein, 1982a) and AHP duration (Burke, 1980)]. We also found motonearon

properties within each motor unit type in self-reinnervated motor units

to be similar to properties in control motor units of the same type.

Besides the above-mentioned relationship between rheobase and input

resistance, other relationships between motoneuron properties in

long-term self-reinnervated motor units are similar to normal MG.

Overall negative correlations were seen between rheobase and input

resistance, and between axonal conduction velocity and input resistance

or AHP half-decay time. As reported by Kuno et al. (1974a,b), fast and

slow motoneurons tend to segregate on plots of conduction velocity vs.

AHP half-decay time (AHP duration in Kuno et al. 1974a,b), in both

control and regenerated motoneurons (Fig. 3-4). Kuno et al. (1974a,b)

defined 'fast' as M3, and 'slow' as soleus motoneurons. We extend this

observation to fast and slow motor unit types within one mixed muscle,

M3, and document the complete recovery of this relationship after nine

months reinnervation. The lack of correlation between these variables

within motor unit types suggests that overall correlations may be a

consequence of the presence of different motor unit types, each with

characteristic properties, as in normal motoneurons (Fleshman a.t al

1981).

Self-reinnervated muscle units did not differ from normal with

respect to speed (isometric twitch time-to-peak, half-relaxation time)

or fatigue resistance (although type FF units were slightly less

fatigable). The exception to complete recovery of overall mean values









for motor unit properties was an overall decline in mean maximum

tetanic tension, especially for type FF units [also seen

in reinnervated FDL (Burke, 1980) and in flexor hallucis longus (Chan &t

Al. 1982)]. This failure to recover tension was due to lack of recovery

of muscle fiber area (type FG fibers). We found no evidence for greatly

enlarged motor units in operated animals (Chan et al. 1982; Dum et al.

1979; Gordon and Stein, 1982a,b). These data suggest that neural

influence may be more effective in regulating muscle twitch speed and

fatigue resistance than muscle unit tension during self-reinnervation

in adult cats.

Twitch speed and fatigue resistance are the parameters which define

motor unit type (Burke et al. 1973). These parameters are also the focus

of most chronic stimulation experiments (reviewed in Pette, 1984;

Salmons and Henrikson, 1981), although Lomo et al., (1980) reported

influence of frequency of activation on force generation by chronically

denervated and stimulated rat soleus muscles. Spector (1984) compared

denervated rat muscles to muscles completely inactivated by TTI, and

reported that while changes in speed of shortening could be accounted

for by activity alone, muscle weight, force generation, and specific

tension appeared to be regulated by release of a trophic substance as

well. Our data are consistent with differential regulation of force and

speed-related muscle properties by the motoneuron.

Relationships between contractile properties after nine months

self-reinnervation were also similar to normal. Motor unit twitch

time-to-peak was negatively correlated with twitch amplitude and with

maximum tetanic tension across the whole population of reinnervated





-46-


units, similar to controls (Bagust and Lewis, 1974). We found no

correlations between contractile properties within motor unit types

(except twitch time-to-peak vs. tetanic tension in type S units).

The similarity between normal and self-regenerated motor units in

motoneuron electrical properties, and in motor unit type distribution,

argues against a selective advantage for any motor unit type in terms of

contacting muscle fibers and surviving, although the number of fibers

innervated by a given unit type may vary (see below). Thus although 10%

of the axons made no functional reconnection with extrafusal muscle

fibers, there was no systematic loss of motoneurons of any type, and the

original proportions of each motor unit type were restored by nine

months self-reinnervation.

The close correspondence between motoneuron "type" and motor unit

type, described above, suggests that proper match-ups between motoneuron

and muscle properties are reestablished at the level of single units,

following long-term self-reinnervation of MG. This can also be seen in

the overall correlations between axonal conduction velocity and twitch

or tetanic tension, axonal conduction velocity and twitch time-to-peak,

and between AHP half-decay time and twitch time-to-peak. This latter

relationship was the strongest between any motoneuron and muscle

property in both normal and operated animals. These correlations are

weak or absent within motor unit types, suggesting that the overall

correlations can be explained by the presence of different types of

motor units in self-reinnervated as well as normal MG.

The AHP half-decay time (or duration) is closely related to the

motoneurons' steady-state firing frequency (Kernell, 1965;






-47-


Oustafsson, 1974). The correlation between AHP half-decay time and

twitch time-to-peak may reflect the control of muscle contractile speed

by pattern and quantity of activation (reviewed in Salmons and

Henrikson, 1981; Pette, 1984). The question still remains as to whether

this match-up is dictated solely by the motoneuron to the muscle fibers

(as suggested by fiber type grouping of units and results from

experiments using chronic electrical stimulation of muscle), or whether

there are also retrograde influences by the muscle on the motoneuron

(Czeh et al. 1978).

Muscle fiber types were present in nearly normal proportions after

long-term self-reinnervation of MS, although there was an increase in

type SO muscle fibers and a corresponding decrease in the proportion of

fast (FG+FOG) muscle fibers in reinnervated MG. "Type grouping" of fiber

types was evident (Dubowitz, 1967; Romanul and Van Der Meulen, 1967).

These data are generally consistent with Chan et al. (1982), Burke

(1980) and Gauthier et al. (1983), who reported restoration of normal

proportions of muscle fiber types with reinnervation. In contrast, Lewis

et al. (1982) presented histochemical data for one cat FHL muscle,

reinnervated for three years, which was 80% type I (equivalent to type

SO) fibers, as compared to a normal 85% type II (FG and FOG). This

result, in combination with their finding of slow units being larger

than fast units in soleus cross-reinnervated by the mixed FDL nerve, led

these authors to propose that slow axons may have a competitive

advantage in reinnervation. Our histochemical data show a slight trend

in the direction of increased numbers of SO fibers, but not of the









magnitude reported by Lewis et al. (1982) and perhaps not outside of

normal variation between animals (real and due to sampling).

The combined data on innervation ratios, muscle fiber type

proportions, and motor unit tension production also suggest that there

could be a slight competitive advantage for type S axons in

reinnervation, as suggested by Lewis et al. (1982). Perhaps within the

type S population, recovery of neuromuscular transmission occurs more

rapidly when the proper match-up between type S motoneuron and SO fiber

is achieved, while fast axons are at a disadvantage unless they

innervate an originally fast muscle fiber. This was suggested by Ip and

Vrbova (1983) for reinnervation of soleus muscle in rats and could

account for the small alteration in motor unit size in the present

study. Ip and Vrbova (1983) showed that either soleus' own nerve or a

foreign nerve were eventually able to innervate soleus muscle

successfully. At early times following the initial surgery, however,

soleus' own nerve induced more tension in the muscle, formed more

synapses with the original endplates, and could follow high-frequency

electrical activation more securely. They noted that the original

post-synaptic specialization remains for some time following

denervation, and may retain its original properties. Finally,

transmission might initially be most effective when the presynaptic

terminal is of the same type as the post-synaptic specialization.

Conclusions

Nine months following self-reinnervation, MN motor units have

properties remarkably similar to normal adult M3. In addition, the

relationships between motoneuron properties and muscle unit properties









are normal, Slow units may have some advantage in making or retaining

contacts with muscle fibers, although there is no strong evidence for

this conclusion. Fiber type grouping and the restoration of motor unit

properties and relationships suggest a major role for the motoneuron in

dictating muscle unit properties. The motoneuron may regulate muscle

unit speed- and fatigue-related properties differently than muscle unit

force generation. The latter property may indicate a limit to

motoneuron regulatory influence. Information concerning the nature of

neural influence on muscle force is of interest with respect to the

observed recruitment of motor units in order of increasing force (Stuart

and Enoka, 1983; Zajac and Faden, 1985) and controversies over the

neural substrate for such a recruitment pattern (Henneman, 1980a; Sypert

and Munson, 1981; Stuart and Enoka, 1983).

This study cannot directly assess whether or not the muscle also

effects motoneuron properties, or whether one type of motoneuron or

another is better able to make early contact with muscle, or recover its

properties more rapidly. Subsequent chapters will address these

questions by utilizing surgical cross-reinnervation of muscles with

different muscle fiber type distributions, and by looking at the time

course of recovery of motoneuron and muscle unit properties during self-

and cross-reinnervation.
















CHAPTER IV
AXOTOMY AND THE TIME COURSE OF SELF-REINNERVATION OF MG


Introduction



CHAPTER III described virtually complete recovery of cat MG

motoneuron electrical properties, muscle unit contractile properties,

and the relationships between them, following nine months

self-reinnervation. One exception was that type FF motor units failed to

regain control tensions, apparently due to a failure of type FG muscle

fiber areas to recover. Based upon proportions of muscle fiber types and

calculated innervation ratios, it was suggested that type S motor units

might have an advantage over type FR units in the ability to make

and/or maintain functional connections with muscle fibers.

Very little is known about motor unit properties during the process

of reinnervation of skeletal muscle because most studies have focused

upon the end-point, when all changes would presumably be completed.

Gordon and Stein (1982a,b) used a chronic paradigm to measure motor unit

twitch tension and axonal action potential amplitude extracellularr)

for cat MG at various times after nerve section and repair. They found

that, at the earliest stages of reinnervation, twitch tensions were low,

a sizable number of axons did not elicit muscle tension, and the normal

relationships between muscle speed and tension, and axonal action

potential amplitude and twitch tension, were lost. When whole muscle

tension reached about 50% of the value eventually attained, the

-50-






-51-


relationships between twitch time-to-peak and tetanic tension returned

(3 mos. post-operative). After this time they saw no further increase

in the estimated number of motor units and suggested that the remaining

increase in whole muscle tension was due to increased muscle fiber

areas. The relationship between axonal action potential amplitude and

twitch time-to-peak returned also (at about three months, although not

at control levels until six months). Gordon and Stein (1982a,b) did not

attempt to classify units by motor unit type until the final acute

experiment, at about one year post-surgery.

Kuno et al. (1974b), studying cat M3 and soleus motoneurons,

reported that at early stages of reinnervation many motoneurons did not

elicit muscle contraction. They reported that these cells did not differ

in electrical properties from motoneurons which did elicit muscle

tension. They suggested that functional reconnection was neither a

necessary nor a sufficient condition for restoration of motoneuron

electrical properties. In contrast, Gordon and Stein (1982b) also

reported that a group of axons did not make functional reconnection with

the muscle, but that in these axons, axonal action potential amplitude

did not recover from axotomized levels.

On the basis of histochemistry of self-reinnervated flexor hallucis

longus (FHL) and a reversed relationship between axonal conduction

velocity and twitch tension in soleus muscle cross-reinnervated by

flexor digitorum longus (FDL) nerve, Lewis et al. (1982) hypothesized

that slow motor units might have a selective advantage in reinnervation

of muscle (see also Ip and Vrbova, 1983).






-52-


The purpose of the present study was to examine the time course of

self-reinnervation of cat M3 to determine: (1) What happens to

motoneuron properties following axotomy? From what level must recovery

occur? (2) Is functional reoonnection with muscle necessary and/or

sufficient for recovery of motoneuron electrical properties? (3) At what

stage are motor unit types first recognizable? How does this relate to

the degree of recovery of motoneuron properties, muscle contractile

properties, and their relationships? (4) Until what stage are new motor

units being formed? (5) Is any motor unit type at advantage in

reinnervating MG? (6) Are there parallels between the time couse of

reinnervation and motor unit ontogeny?

Results

Whole Muscle

Table 4-1 lists values for muscle wet weights and whole muscle

twitches for all treatment groups where muscle twitch tension could be

elicited by nerve stimulation. After the M3 nerve was severed there was

muscle atrophy. At the earliest stage of reinnervation examined (low-re;

two cats), muscle weight and tension were less than half of control

values. At this stage isometric twitch time-to-peak was prolcnrfed, as

was half-rise time and half-relaxation time.

The medium self-reinnnervation (med-re; two cats) category was

defined in terms of recovery of tension. These MG muscles had maximum

twitch tensions of over one half of nine month self-reinnervated





-53-


Table 4-1. Whole Muscle Properties: Time Course of Self-Reinnervation. a


NORMAL LOW-RE ED-RE LONG-RE


Twitch Time-To-Peak b (ms) 321 46114 340 333
Twitch Half-Rise Time b (ms) 11+0 193 130 100
Twitch Half-Relaxation Time b (ms) 22-2 702 388 252
Twitch Tension b (g-wt.) 2123151 7737 89311 1609276
Muscle Weight (g) 9.01 5.00 4.4.1 7.01
Muscle Wt./Cat Wt. (g/kg) 3.00 1.60 1.50 2.30
Twitch Tension/Cat Weight (g-wt./kg) 74265 24+11 30219 48866


a. MeansSE (for NORMAL n=14; for LOW-RE n=2; for MED-RE n= 2;
for LONG-RE n=4).
b. Twitch at muscle length at which maximum tension was obtained.









(long-re) values (maximum twitch >1000g-wt tension). This group was also

characterized by time after the initial surgery (63 and 71 days). By

this stage values for contraction time (time-to-peak, half-rise,

half-relaxation) had recovered to normal or near normal levels. Muscle

weights were about 60% of long-re (significant at aLPHA = 0.001). Data

for the nine months reinnervated and normal MG are from CHAPTER III.

One animal (FE17), for which we only have whole muscle contractile

and histochemical data, was intermediate between the low-re and med-re

groups in terms of recovery of tension. This animal was sacrificed at 52

days post-surgery. Maximum whole muscle twitch was 281 g-wt.and

time-to-peak was 6 ms. Muscle weight was 5.5g and estimated

physiological cross-section was 2.1 cm2.

A second animal (FE12) also exhibited recovery to a level

intermediate between the low-re and med-re stages after only 34 days.

This animal's whole muscle twitch was closer to the med-re level

(maximum twitch of 653 g-wt., 30 ms time-to-peak). The mass of M3 was

7.4g and physiological cross-section was estimated at 3.1 cm2. Motor

unit data were obtained from this animal.

These two animals indicate that recovery was a continuous process,

with considerable variability in the rate of recovery for different

animals. Data obtained from the other animals were generally consistent

at a given defined stage.







-55-


Axotomy

To examine the physiological effects of axotomy upon HG motoneurons,

four animals were examined three to five weeks (25, 26, 33 and 35 days)

following nerve section. No tension could be elicited from the MG muscle

by MG nerve stimulation, nor were electomyographic responses seen.

Although it is not known whether there were connections which were too

weak to elicit tension, structural regeneration of the nerve terminals

is not necessarily immediately accompanied by functional neural

transmission (Birks et al. 1960; but see Carmignoto et al. 1983).

The mean values for motoneuron electrical properties are seen in

Table 4-2. Frequency histograms for motoneuron electrical properties

following axotomy are seen in Figures 4-1 and 4-2. There was a marked

decrease in axonal conduction velocity in axotomized motoneurons (Ecoles

et al. 1958; Kuno et al. 1974a,b; Hoffer et al. 1979; Milner and Stein

1981; Cragg and Thomas, 1961; Mendell et al. 1976; Kiraly and Krnjevic,

1959; Gallego et al. 1979b). The range of values for axonal conduction

velocity was similar to control, although shifted to lower values (Fig.

4-2B). This decreased conduction velocity is believed to be due

primarily to decreased axonal diameter (Gutman and Sanders, 1943;

Sanders and Young, 1946), as well as shorter internodal distance and

altered ion channel distribution (Beeryet al. 1944; Cragg and Thomas,

1964; Ritchie, 1982; Kocsis et al. 1982; Kocsis and Waxman, 1983).

Myelin thickness does not change markedly with axotomy (Gillespie and

Stein, 1982).






-56-


Table 4-2. Motoneuron Electrical Properties: Axotomy and Time Course of
Self-reinnervation.a,b,c


FF FI FR S ALL
RHEOBASE (nA)


201 (70)


293 (3)
24+1 (33)


161 (6) 111 (37)


162 (9) 112 (13)
183 (3) 111 (18)


50 (34) 141 (147)+
40 (67)*
51 (35)'
31 (8) 101 (33)'
61 (19) 151 (73)+


INPUT RESISTANCE ( :hms)


0.60 (56)


0.70 (3)
0.70 (22)


0.90 (3)


0.8+0
0.7+0


1.11 (30) 1.50 (28)


1.70 (12)' 1.90 (7)
1.20 (10) 1.30 (11)


1.00 (117)+
2.60 (60)0
2.41 (35)'
1.80 (29)'+
1.00 (45)+


RHEOBASE/INPUT RESISTANCE (nA/Mohms)


1+1 (30)


92 (12)
102 (10)


41t (28) 212 (117)+
20 (59)*
30 (35)'
42 (7) 123 (29)'+
61 (11) 233 (45)+


AHF HALF-tDF'IY T"T? (ur-


192 (5) 251 (31)


161 (9) 232 (12)
231 (3) 242 (18)


49_3 (28)


433 (8)
44+3 (19)


281 (118)
301 (65)
272 (38)
271 (32)
271 (73)


AXO'IAL CC;PU'TIQON VELO:iTY fm1 -


971 (55) 1004 (7)


821 (10)'
945 (3)


992 (25)


812 (24)


801 (14)0 751 (9)'
962 (18) 812 (19)


941 (111)+
701 (72)'
6t1 (36)'
74+2 (36)*
901 (73)+


a. Means + SE (number of units).
b. = Significant difference from NORMAL (p<0.01);
c. + = Significant difference from NO-RE (p<0.01);


NORMAL MG
NO-RE
LOW-RE
MED-RE
LONG-RE


NORMAL MG
NO-RE
LOW-RE
MED-RE
LONG-RE


NORMAL MG
NO-RE
LOW-RE
MED-RE
LONG-RE


352 (55)


5218 (3)
404 (22)


23.5 (3)


224 (7)
3312 (2)


NORMAL M;
NO-RE
LOW-RE
MED-RE
LONG-RE


221 (54)


131 (3)
201 (33)


NORMAL MG
NO-RE
LOW-RE
MED-RE
LONG-RE


852 (3)
962 (33)






-57-


There was no change in mean AHP half-decay time following axotomy

(Gustafsson, 1979; Kuno et al. 1974b), but the range was compressed from

both extremes (Fig. 4-2A). In contrast, Gustafsson and Pinter (1984b)

reported a slight but significant increase in AHP duration for

lumbosacral motoneurons (16 of which were triceps surae). Kuno et al.

(1974a) also showed a slight increase in MG AHP durations following

axotomy. This later study also examined the slow soleus motoneurons,

which exhibited markedly decreased AHP durations following axotomy. This

finding, in conjunction with the altered distribution of AHP durations,

suggests that the difference between studies reflects the relative

composition of fast and slow motor units in the motoneuron pool

examined. The presence of approximately 25% slow units in MG may offset

the change in AHP duration of fast units (Kuno et al. 1974a; Gustafsson

and Pinter, 1984). Gustafsson and Pinter (1984) suggested that the

compressed range for AHP duration with axotomy indicated that fast

motoneurons increase AHP duration, while slow motoneurons decrease AHP

duration. Alteration in AHP half-decay time could reflect changes in the

kinetics of the Ca++-dependent K+ conductance thought to underlie the

AHP (Barrett et al. 1980; Krnjevick et al. 1978) or changes in the

magnitude of membrane nonlinearities (Ito and Oshima, 1965; Gustafsson

and Pinter, 1985).

Most studies on spinal motoneurons have reported an increase in

input resistance with axotomy (Gustafsson, 1979; Gustafsson and Pinter

1984). In contrast, Kuno and Llinas (1970) reported no change of input

resistance following nerve section. The discrepancy may be due in part,

to different criteria for recognizing axotomized cells.





-58-


I ____
S t; 5E :.2a;


:h; ES S'L; .Y *


Figure 4-1. Frequency histograms for MG motoneuron properties following
section of the MG nerve (filled), compared to normal MG motoneurons
(unfilled). (A) Rheobase, note decreased range and mean vs. controls.
(B) Input resistance, note similar range, but shifted to higher values
than controls. (3) Ahp half-decay time, note range compressed from both
extremes, no change in means. (D) axonal conduction velocity, range is
decreased and mean is lower.






-59-


Kuno and Llinas (1970) included only those cells exhibiting a 'partial'

active dendritic response to afferent input, in their axotomized

population. Also, in that study motoneurons were axotomized by ventral

root section, as opposed to peripheral nerve section in the present work

(also Gustaffson, 1979; Gustafsson and Pinter, 1984). The axon reaction

is known to be dependent upon the length of the intact proximal stump

(Kuwada and Wine; 1981, Lieberman, 1971; Mendell et al. 1976). The

range of values for input resistance was similar to that of normal MG,

although shifted to higher values (Fig. 4-1B). Mechanisms thought

responsible for alterations of input resistance with axotomy are

discussed by Gustafsson and Pinter (1984).

Rheobase was decreased in axotomized motoneurons, in agreement with

earlier reports (Eccles et al. 1958; Kuno and Llinas, 1970; and

Gustafsson, 1979). The range of values seen for rheobase was compressed

as well as shifted to lower values (Fig. 4-1A). Presumably the

decreased rheobase indicates altered excitability of the initial segment

and the soma-dendritic membrane (Eccles et al. 1958; Faber, 1984).

The relationship between rheobase and input resistance is altered by

axotomy (Fig. 4-3A). The range of values was compressed. The mean value

for rheobase/input resistance was lower than normal (Table 4-2).

When compared to motoneurons from normal type S units, axotomized MG

motoneurons had significantly higher input resistance, slower axonal

conduction velocity and lower rheobase/input resistance. Thus, while

some axotomized cells fell within the type S range for some parameters,

they were not identical in properties to normal type S motoneurons.






-60-


A B
kn 3S 35

a Ii--l "^ -
I- 2S -
0I 5I


0 15 0 is


zs

29 48 6 8 18N 5s 73 9 lit 1 IN
RHP HALF-DECAY TIME (ms) RXONRL CONDUCTION VELOCITY (m/s)












Figure 4-2. Frequency histograms for MG motoneuron properties following
section of the MG nerve (filled), compared to normal MG motoneurons
(unfilled). (A) AHP half-decay time, note range compressed from both
extremes, no change in means. (B) axonal conduction velocity, range is
decreased and mean is lower.






-61-


In the no-re population, the correlation seen in control motoneurons

between rheobase and input resistance was lost (Fig. 4-3A), as was that

between AHP half-decay time and axonal conduction velocity (Fig. 4-4A).

There was no segregation of motoneurons with respect to these

properties. For AHP half-decay time vs. axonal conduction velocity, 85%

(51/60) of motoneurons sampled were outside the normal range for either

fast or slow motoneurons. Motoneuron types based on the ratio rheobase:

input resistance and AHP half-decay time (Zengel et al. 1985) could not

be recognized in the no-re population.

These data are consistent with a dedifferentiationn' of motoneuron

properties from the normal adult state (Kuno et al. 1974a; Gustafsson

and Pinter, 1984). While some properties of axotomized motoneurons are

similar to those of normal type S motoneurons (rheobase, input

resistance, rheobase/input resistance), AHP half-decay time is shorter,

and axonal conduction velocity is slower than in normal type S units.

Thus, the dedifferentiated state is not simply an 'S' or 'super S'

motoneuron type (Gustafsson and Pinter, 1984).

Early Reinnervation

We examined two animals (39 and 40 days post-operative: low-re)

which had MG muscles which generated 41 and 115 g-wt. twitch tension,

respectively, in response to MG nerve stimulation. Previous studies

indicate that a minimum of about four weeks is required before muscle

contraction can be elicted by nerve stimulation (Eccles et al. 1962;

Gordon and Stein, 1982b).









































A


a i r i 1CF:
r Ii I, a ar i


-62-


C _


- 3-


t
:i
i:
~----
,? s-:


-I

*.*


SI

a- *


* Li U aa L I 1 I I
I~u UsiKuctr,


Figure 4-3. Relationships between rheobase and itput resistance
following section of the MG nerve, and at early and intermediate times
of reinnervation. (A) axotomy, note uniformity of the sample and
altered distribution (N= non-contract). (B) low-re, distribution is
similar to no-re stage; no differences between contracts (C) and
non-contracts (N). (C) med-re, note recstablishment of sr-egation by
motor unit type, although distribution is not at normal levels.






-63-


Of 37 motoneurons sampled, 20 elicited muscle unit contraction

(57%). Assuming 280 motoneurons in the normal M1 motor nucleus (Boyd and

Davey, 1968; Burke et al. 1977), this suggests that approximately 160

axons innervated MG muscle.

Motoneuron electrical properties. Low-re motoneurons had overall

mean values which were significantly different from controls for all

motoneuron electrical properties except AHP half-decay time (Table

4-2). In most cases the overall means were indistinguishable from the

no-re population. There were no significant correlations between any

motoneuron parameters at the low-re stage, unlike controls, but similar

to axotomy (Figs. 4-3B, 4-4B).

Motoneurons at the low reinnervation stage were divided into two

groups on the basis of whether they elicited muscle contraction

(contracts) or not (non-contracts). The only differences in mean values

between these two groups were rheobase and input resistance (both lower

in contracts; Table 4-3). The similarity between properties of contracts

and non-contracts could be interpreted in two ways: either making

functional reconnection is not sufficient for re-establishment of normal

motoneuron properties, or additional time is required following

reinnervation for normal electrical properties to be expressed.

Kuno t1 aL (1974b) found two MG motoneurons (five months

self-reinnervated muscle) which did not elicit muscle tension, but did

not differ from connected cells in AHP duration, axonal conduction















































4'4
O.


r
FI.


u
j

i i,


r
r-

t
r-
P
B


Pt

4 *[
S( *p. Cf [


axA ON L t.
B CO )iDK lD'. t.IXXlt1. 1


4*- J* p. ---- 0 0 40.__~.i.


Figure 4-4. Relationships between AHP half-decay time and axonal

conduction velocity after nerve section, and at early and intermediate
stages of reinnervation. (A) no-re. Virtually all motoneurons fall
outside the normal range. (B) low-re. Virtually all motoneurons fall
outside the normal range. There was no difference between contracts (C)

and non-contracts (N). (C) med-re. Many contracts (F,I,R,or S) approach
or enter the range of control motoneurons, while all non-contracts (N)
fall outside the normal range. There was segregation of fast and slow

motoneurons. Solid lines in all graphs outline the distribution of slow

(upper) and fast motoneurons in controls.


* V


V






-65-


velocity, or action potential amplitude. They preferred the explanation

that functional reoonnection is neither necessary nor sufficient for

recovery of motoneuron properties.

That functional reconnection is necessary for re-establishment of

normal electrical properties is suggested by the similarity between

non-contracts at all times following axotomy (Table 4-4). At the med-re

and long-re stages non-contracts had mean values significantly different

from the overall sample at the same stage. Mean values for rheobase and

rheobase/input resistance (med-re only), input resistance, and axonal

conduction velocity also differed from type S units at the same stage of

recovery. AHP half-decay times were short, as in axotomy, and different

from control type S units. These data are consistent with suggestions,

based on recording extracellular action potentials of single units, that

the effects of peripheral axotomy are not continuous, but reach a

stable, plateau value (Davis et al. 1978). This suggests that motoneuron

survival following peripheral axotomy is not dependent upon connection

to muscle in regenerating adult motoneurons, although expression of

normal electrical properties is. All cells at the long-re or med-re

stages which made functional reconnection with the MG muscle showed

recovery of motoneuron properties, suggesting that reconnection, with

sufficient recovery time, is sufficient for expression of mature

electrical properties.

There were some non-contracts in the long-re and med-re populations

with properties in the normal range for one or several motoneuron

properties. These cells could have been injured in the final dissection

or perhaps represent alpha-fusimotor innervation (Gregory et al. 1982).






-66-


Table 4-3. LOW-RE Motoneuron Electrical Properties: Contract vs.
Non-Contract. ac


CONTRACTS


RHEOBASE (nA)
RN b (Mohms)
RHEOBASE/RN
AHP HALF-DECAY TIME (ms)
AXONAL C.V. (m/s)


41
1.50
30
272
66+2


a. Means SE (number of cells).
b. RN = input resistance.
c. = significant difference fr
(alpha = 0.01).
d. C.V. = conduction velocity.


(20)1
(20)"1
(19)
(20)
(20)


NON-CONTRACTS


61
2.30
31
282
612


(17)
(15)
(15)
(17)
(17)


om NON-CONTRACT (alpha = 0.05);


Table 4-4. Motoneuron Electrical Properties of Non-contracts: Self-
reinnervation Model. a,c


NO-RE


RHEOBASE (nA)
RN b (Mohms)
RHEOBASE/RN
AHP BALF-DECAY TIME
AIONAL C.V. (m/s)


(ms)


30
2+0
301
701


LOW-RE


(67)
(60)
(59)
(65)
(72)


6_+1
2+0
31
28+2
61+2


MED-RE


(17)'
(15)
(15)
(17)
(17)


30
30

302
56_+3


(11)
(12)
(11)
(12)
(12)*


a. Means SE (number of cells).
b. RN = Input resistance.
c. = significant difference from NO-RE (alpha = 0.05);
0" (alpha = 0.01).
d. C.V. = conduction velocity.


LONG-RE


71
20
5-4
304
64t7


(8)
(5)
(5)*
(8)
(8)


--- "" ""~ "'






-67-


At the long-re stage the vast majority of non-contracts were outside of

the normal range for AHP half-decay time vs. axonal conduction velocity

(o.f. Fig. 3-4).

Muscle unit contractile properties. All speed-related properties of

the whole muscle twitch were prolonged at the low-re stage (Table 4-1;

twitch time-to-peak, half-relaxation time, half-rise time). Prolonged

isometric time-to-peak has been reported to occur with denervation (Kean

t al. 1974; Spector, 1984), with relaxation prolonged more than twitch

rise-time. This has been attributed to alterations in the sarooplasmic

reticulum, troponins, and myosin in the denervated muscle fibers (Cecchi

et al. 1984; Spector, 1984). At the low-re stage, motor units did not

fit into adult motor unit types and so were treated as a single

population. The mean value for low-re units' potentiated twitch

time-to-peak was not significantly different from control (Table 4-5).

Half-relaxation time was prolonged in low-re units. Amplitudes of

potentiated twitch and maximum tetanus were greatly reduced in low-re

units. All low-re units were fatiguable, but most showed residual

tension at the end of the fatigue test.

We found no significant correlations between motor unit contractile

properties in the low-re population. The relationship between twitch

time-to-peak and maximum tetanus (-0.54, p<0.07)) approached

significance.

These results are in agreement with Gordon and Stein (1982a,b) in

that initially after reinnervation mean unit tensions were smaller,

with no correlations between motor unit speed and tension.






-68-


Table 4-5. Muscle Unit Contractile Properties: Time Course of
Self-Reinnervation.1,2

FF FE FR S ALL

TWITCH TIME-TO-PEAK d(ms)


291 (56)

302 (3)
281 (32)


282 (7) 261 (30)


28t2 (10)
255 (3)


301 (14)
271 (17)


584 (22)

59t (9)
55+3 (18)


3412 (115)
382 (12)
373 (36)
342 (70)


TWITCH TENSION d(g-wt.)


181 (56)

191 (3)
10+1 (32)*


41 (7) 10 (28) 0.20 (22)


62 (10) 10 (13)
2.6 (3) 10 (17)


0.40 (9)
1.00 (18)


91 (113)
00 (12)9
41 (35)"
51 (70)*


TWITCH HALF-RELAXATION TIME d(ms)


272 (7) 261 (29)


29-2 (10)
245 (3)


312 (13)
252 (16)


74S8 (19)

699 (9)
514 (15)


332 (111)
44t5 (12)
40t4 (35)
322 (66)


MAXIMUM TETANIC


613 (84)

767 (3)
3T4 (31)*


20+3 (7) 13i1 (45)


25.6 (10)
200 (3)


92 (14)
133 (17)


71 (39) 352 (175)
41 (23)'
4.+1 (9) 163 (36)*
6-1 (16) 232 (67)*


FATIGUE INDEX


0.00 (82) 0.50 (7)

0.1+0 (3) 0.50 (10)
0.10 (33)* 0.50 (3)


1.00 (45)

0.90 (13)
1.20 (17)


1.10 (40)

1.00o (8)
1.10 (13)


0.60 (174)
0.20 (11)
0.7 0 (34)*
0.6+-0 (63)


Means + SE (number of units).
* = Significant difference from NOERAL MG (p<0.01).
Potentiated twitch.


NORMAL MG
LOW-RE
MED-RE
LONG-RE


NORMAL MG
LOW-RE
MED-RE
LONG-RE


NORMAL MG
LOW-RE
MED-RE
LONG-RE


241 (56)

306 (3)
282 (32)


NORMAL MG
LOW-rF
MED-RE
LONG-RE


NORMAL MG
LOW-RE
MED-RE
LONG-RE


TENSION (g-wt.)






-69-


Muscle histoohemistry. Low-re MO contained 50% type FG muscle

fibers, 28% type FOG, and 23% type SO, similar to controls (Table 4-7).

It is uncertain to what extent this reflects the original innervation of

the muscle fibers, as fiber type grouping (Dubowitz, 1967; Romanul and

Van Der Meulen, 1967; Karpati and Engel, 1968a) was not seen at this

stage. Mean muscle fiber area was decreased at the low-re stage, with

fast fibers (types FG and FOG) affected more than type SO fibers (Table

4-7).

The muscles from the animals intermediate in recovery between low-re

and med-re stages contained 55% type FG, 17% type FOG, and 28% type SO

fibers (FE12) and 44% type FG, 32% type FOG, and 25% type SO fibers

(FE17). Of 12 motor units sampled in cat FE12, six were type FF, one

was type FR, three were type S motor units, and two were non-contracts.

No motor unit data were obtained from FE17.



Relationships between motoneuron electrical properties and muscle unit

contractile properties.. We found no significant correlation between

any combination of motoneuron and muscle properties at the low-re stage

(Gordon and Stein, 1982b). At two months following the initial surgery,

Gordon and Stein (1982a,b) reported no correlation between action

potential amplitude (correlated with axonal conduction velocity) and

twitch tension, or between twitch contraction time and twitch tension.






-70-


Medium Reinnervation Stage

Two animals were examined which generated greater than 50S of

long-re whole muscle twitch tension (63 and 71 days; med-re). At this

stage motor unit types could be distinguished by contractile criteria.

The proportion of fast and slow units at the med-re stage was similar to

normal MG (75% fast, 25% slow vs. 76:24 in normal HM; 69:31 in long-re

MG). There were 8% type FF, 28% type FI, 39% type FR and 25% type S

units (controls: 48:4:24:24; long-re: 50:4:15:31; Table 7). The

increased proportion of type FI units appears to be at the expense of

type FF units. This may indicate a general increase in fatigue

resistance of fast units at this stage, perhaps due to previous

polyneuronal innervation, increased recruitment due to lowered overall

force-generating ability of the muscle, or a general increase in

oxidative enzymes as a function of reinnervation.

Statistical differences between means for motor unit parameters at

the med-re stage were similar to normal and long-re motor units (Table

4-6).

Motoneuron electrical properties. The overall means for med-re

motoneuron electrical properties show a definite shift towards those of

the long-re and normal populations (Table 4-2), although axonal

conduction velocity, rheobase/input resistance and rheobase were still

lower, and input resistance was still higher in med-re than long-re or

normal motoneurons (Table 4-2). Mean AHP half-decay time was

essentially the same at all stages.






-71-


Table 4-6. Results of Tukey's Studentized Range Test: Significance of
Differences Between Motor Unit Types for MED-RE Motor Units.


MED-RE


Axonal Conduction Velocity
Rheobase
Input Resistance
Rheobase/Input Resistance
AHP Half-Decay Tipe
Twitch Tension a'
Twitch Time-To-Peak b
Twitch HRT b',
Maximum Tetanic Tension a


a. The same result was obtained for raw
body weight.
b. Potentiated twitch.
c. HRT = half-relaxation time.


NS
F>I>(R,S)
NS
F>(I,R,S)
NS
F>I>(R,S)
(F,I,R) (F,I,R) F>I>(R,S)


data and data normalized for


0.05
0.01
0.05
0.01
0.05
0.01
0.01
0.01
0.01






-72-


Mean values by motor unit type, for motoneuron electrical

properties, were generally similar to properties of the same type of

motoneuron in normal or long-re MG motoneurons. Only input resistance

(type FR) and axonal conduction velocity (types FF and FR) differed

between long-re and med-re motoneurons of the same type (Table 4-2).

Fig. 4-3C shows the relationship between rheobase and input

resistance for med-re motoneurons. Note that segregation by motor unit

type is re-established at this stage, although lower rheobases and higher

input resistances tend to alter the distribution from normal. The

correlation between log rheobase and input resistance was -0.80

(p<0.0001). Within unit types log rheobase and input resistance were

correlated for type S units only (-0.80, p<0.03)).

AHP half-decay time was negatively correlated with axonal conduction

velocity across all units (r= -0.50, p<0.0001; Fig. 4-4C), as in normal

and long-re motoneurons (Fig. 3-2). This relationship was significant

within type FR units (-0.79, p<0.002), but not type FF or type S units.

There was some segregation into fast and slow groups by the relationship

AHP half-decay time: axonal conduction velocity (Fig. 4-4B), although

this relationship was not as in normal or long-re motoneurons (Fig 3-4).

Five of eight type S motoneurons were within the normal range for this

relationship (63%), as were 10 of 22 fast motoneurons (45%). All

non-contracts (12/12) fell outside the normal range for either fast or

slow motoneurons.

Motoneuron type was determined according to the criteria of Zengel

et al. (1985; AHP half-decay time >30ms = fast, AHP <30ms = slow; type

FF units have rheobase/input resistance >18, type S units <7, and type






-73-


FR units between 7 and 18). At the med-re stage, there was an 82%

agreement (18/22) between electrically determined motoneuron type, and

motor unit type determined from contractile properties. AHP half-decay

time predicted fast and slow motor units in 91% of cases (29/32).

Muscle unit contractile Droperties. At this stage certain

contractile properties have recovered more than others (Table 4-5). The

overall means for twitch time-to-peak, half-rise time, and

half-relaxation time were similar to normal. Twitch tension (raw data

and normalized by body weight) recovered nearly to the level shown by

long-re units (smaller than control values). This was also true of

maximum tetanus (raw and normalized). Overall fatigue index was higher

than for normal or long-re units, reflecting the increased proportion of

types FR and FI units. All units displayed some residual tension after

the fatigue test.

Across the whole population, twitch time-to-peak was significantly

correlated with the log of twitch amplitude/body weight (r= -0.32,

p<0.04) and tetanic tension/body weight (r= -0.58, p<0.0001), as in

normal and long-re units (CHAPTER III). These relationships did not

hold within any motor unit type.

Relationships between motoneuron electrical properties and muscle

urlt sc. tr.ctile tLrorertie As noted above, there was good agreement

between motoneuron type and motor unit type at the med-re stage. Most

correlations found between motoneuron and muscle properties in normal

and lonr self-reinnervated MG had recovered by the med-re stage. Across

all units, AHP half-decay time was positively correlated with twitch

time-to-peak (0.82, p< 0.0001). This relationship was not significant






-74-


within motor unit types. Axonal conduction velocity was correlated with

twitch time-to-peak (-0.51, p<0.0001), the log of twitch tension/body

weight (0.43, p<0.01), and log maximum unit tetanic tension/body weight

(0.63, p<0.0001). The relationship with time-to-peak was only

significant within FR units (-0.73, p<0.03) and the relationship with

log tetanic tension/body weight within FR units (0.59, p<0.02). There

were no significant correlations between axonal conduction velocity and

log twitch tension/body weight within motor unit types.

Gordon and Stein (1982a,b) noted a parallel recovery of axonal

action potential amplitude and motor unit twitch tension. In their

study, units recovered normal tension five to six months following

surgery (nerve-nerve suture). The normal positive correlation across the

whole population of units, for action potential amplitude and twitch

tension, was lost in early reinnervation, and did not recover until the

sixth postoperative month (Gordon and Stein, 1982b). The normal negative

correlation between twitch contraction time and tension recovered

earlier, at three months. These authors felt that the early lack of

correlation between axonal action potential amplitude and twitch tension

may be explained by motor units being heterogeneous with respect to

metabolic enzymes at early stages. They suggested that recovery of

normal relationships between nerve and muscle depends upon time and

extent of whole muscle recovery, with whole muscle recovery having the

best predictive value.

Muscle histochemistry, muscle fiber areas. and innervation ratios.

Histochemistry did not indicate the same magnitude of shift towards

fatigue-resistant units as did the motor unit type distribution. At this






-75-


stage 43% of the muscle fibers were type FO, 24% type FOG, and 33% type

SO (Table 4-7). The med-re stage was the first one in which fiber-type

grouping (Dubowitz, 1967) of muscle fibers was seen. The overall

distribution was significantly different from controls (Chi-square, 95%

confidence level). Comparison to expectations for an assumed

hypergeometric distribution showed no significant differences for any

individual muscle fiber type. Oxidative fibers (FOG+SO) made up 57% of

the population, as compared to 44% in controls (31% in long

self-reinnervated). This change is in the direction suggested by the

motor unit type distribution. Perhaps the qualitative comparison of NADH

staining intensity is insufficiently sensitive to detect the degree of

changes in enzyme levels, or the relationship between NADH staining and

fatigue resistance is not particularly strong (Baldwin et al. 1984).

We estimate physiological cross-section at 2.4cm2. Mean fiber areas

and relative innervation ratios are found in Table 4-7. Mean fiber areas

show recovery towards normal values for types FG and FOG fibers, while

type SO fibers are smaller than controls. (see CHAPTER III).

With denervation, types FG and FOG fibers atrophy to a greater

extent than type SO fibers (low-re stage; see also Lowrie and Vrbova,

1984). With time, type SO muscle fibers decrease in size until the

med-re stage, then recover to normal levels. Type FOG fibers recover to

normal size earlier, at the med-re stage. Type FG fibers, on the other

hand, never recover to control levels, although type FG fibers remain

the largest. Motor unit tension recovery follows the pattern of

recovery of muscle fiber area (see below).






-76-


Table 4-7. Motor Unit Types, Musole Fiber Types, and Innervation Ratios:
Time Course of Self-Reinnervation. 6


MUSCLE FIBER TYPES

I Histochemical Composition
FG FOG SO n/N a
NORMAL MG 56 23 21 4200/7
LOW-RE 50 28 23 900/2
MED-RE 43 24 33 900/2
LONG-RE 54 15 31 3000/5

Observed Mean Fiber Area (um2)
ALL
NORMAL MG 3873 2264 1972 3104
LOW-RE 1439 1274 1803 1491
MED-RE 2125 1634 1211 1714
LONG-RE 3098 2355 2296 2738

EQUIVALENT MOTOR UNIT TYPE

I Of Population
FF+FI FR S n/N a

NORMAL MG 48+4 24 24 176/16
MED-RE 8+28 39 25 36/2
LONG-RE 46+4 24 26 72/4

Est. Number in Pool d
AtLb
NORMAL MG 134+11 67 67 280
LOW-RE X X X 160
MED-RE 2+59 82 53 210
LONG-RE 116+10 61 66 252

CALCULATED VALUES

FF+FI FR S
Relative IR c

NORMAL MS 1.1 1.0 0.9
MED-RE 1.2 0.6 1.3
LONG-RE 1.1 0.6 1.2


a. Number of Cells/Number of Animals.
b. [280 (280)(% non-contracts)]
c. Relative Innervation Ratio = %Muscle Fiber Type/% Motor Unit Type.
d. Estimated as (Total number of motoneurons)(% motor unit type).
e. Motor unit types could not be distinguished at the LOW-RE stage.







-77-


Relative innervation ratios indicate a possible disadvantage of type

FR motoneurons and advantage of type S motoneurons in capturing muscle

fibers. At the long-re stage there is an even greater trend towards low

relative innervation ratio of type FR units and increased ratio in

type S units than observed at the long-re stage (CHAPTER III).

Gordon and Stein (1982a,b) used chronic recording techniques to

follow the time course of self-reinnervation of cat MG. They calculated

that the estimated number of units did not change from early

reinnervation to the plateau of recovery although whole muscle and

single unit tensions increased (see also Kuno et al. 1974a). These

authors speculated that the primary change was an increase in fiber

diameter. Our data support the notion that a large proportion of the

increase in muscle tension from the med-re stage to the long

self-reinnervated stage is due to increase in fiber area (160$ increase

in mean fiber area, 120% increase in whole muscle twitch tension, 129%

increase in mean motor unit tetanic tension). After nine months

reinnervation, all three of these parameters were still below normal

values (88%, 78%, 63% of normal, respectively).

We saw a larger proportion of non-contracts at the med-re

stage, relative to nine-month reinnervated MG (25% vs. 9%). We estimated

the number of innervating axons from the percentage of non-contracts

(12/48 = 25%) as 210. This indicates that a proportion of axons, which

eventually form functional connections with muscle fibers, had not yet

done so at 63 or 71 days. Alternatively, a greater percentage of units

at this stage which are connected do not elicit measurable levels of

tension.






-78-


At the med-re stage we found significant correlations between

contractile parameters, as well as between axonal conduction velocity or

AHP half-decay time and contractile parameters. It appeared however,

that some axons destined to do so had not made functional connections at

this time (63-71 days). In general, recovery of muscle and motor unit

properties occurred in parallel.

Discussion

This study examined the influence of functional connection with

muscle on expression of motoneuron electrical properties. Also examined

was the time course of recovery of motor unit properties during

self-reinnervation of the cat MG muscle. The purpose was to determine

the level from which motoneurons must recover, and the progression of

recovery, in order to place the long-term reinnervation results in

perspective. In addition, such data were required for interpretation of

ongoing studies of reinnervation of foreign muscles by motoneurons.

Following nine months recovery from nerve section and self-reunion,

most motoneuron and muscle unit properties recover to control levels,

and normal relationships between motoneuron and muscle properties are

re-established (Eagust et al. 1981; Lewis et al. 1982; Chan et al. 1982;

Burke, 1980; CHAPTER III). In this study we examined motoneuron

electrical properties and the contractile properties of the respective

muscle units, after intermediate periods of recovery from nerve section.

Following section of the M3 nerve, MG motoneurons become more

excitable to somatic current injection (decreased rheobase), depolarize

more in response to a given current input (increased input resistance),

and M3 axons conduct impulses at a lower rate. Axotomized motoneurons






-79-


did not segregate on the basis of either the ratio rheobase: input

resistance or the relationship of AHP half-decay time and axonal

conduction velocity. All of these data support the notion that following

axotomy, motoneurons 'dedifferentiate' (Kuno et al. 1974a; Gustafsson

and Pinter, 1984). Dedifferentiation can be regarded as a shift to a

growth state for the motoneuron (Watson, 1976).

Huiszar et al. (1975) noted the resemblance between axotomized

motoneurons and those of kittens, and suggested that this

dedifferentiation returns adult motoneurons to a state similar to early

ontogeny. Gustafsson and Pinter (1984a) suggested that axotomized

motoneurons dedifferentiate to a level similar to that of adult slow

motor units. The present data show that while many electrical properties

of axotomized motoneurons overlap the range of type S motoneurons, in

most cases the mean values for axotomy are different, and certain

parameters (AHP half-decay time, axonal conduction velocity) are very

different in axotomized motoneurons than in normal type S motoneurons.

In addition, the relationship between AHP half-decay time and axonal

conduction velocity was different for axotomized motoneurons than for

type S motoneurons. Thus type S motoneurons, as well as fast

motoneurons, represent a differentiatiated state from axotomized or

immature motoneurons.

Functional connection to muscle and expression of motoneuron electrical
properties.

One issue we addressed was whether functional reconnection of

motoneuron to muscle fibers was necessary or sufficient for recovery of






-80-


motoneuron electrical properties. Kuno et al. (1974b) measured axonal

conduction velocity, action potential overshoot amplitude, resting

membrane potential, and duration of the AHP at various times (up to five

months) following self-reinnervation of cat MG and soleus. At early

times, with incomplete reinnervation, they reported no difference

between properties of those motoneurons which elicted muscular

contraction, and those that did not (both groups appeared axotomized).

They interpreted this as evidence that functional reconnection is not

sufficient for recovery of normal motoneuron properties. Kuno et al.

(1974b) reported two of 25 M3 motoneurons which did not elicit muscle

tension following five months recovery, but had AHP duration and axonal

conduction velocity in the normal range. Their interpretation was that

functional reconnection was not necessary for recovery of motoneuron

properties. One potential complication bearing on this interpretation

is that properties of axotomized motoneurons overlap somewhat with

normal type S motor units in terms of electrical properties (Gustafsson

and Pinter, 1984; this study). In the Kuno et al. (1974b) study, all MG

motoneurons were regarded as 'fast', and all soleus motoneurons as

'slow'. Normal MG contains approximately 25% type S motor units (Burke

et al. 1973; Burke, 1981; Fleshman et al. 1981; CHAPTER III).

Our data show that motoneurons which do not make functional

reconnection with muscle display membrane electrical properties similar

to axotomized motoneurons. At the low-re stage, electrical properties of

motoneurons with functional reconnections, and those of non-contracts

were not different from one another, and neither differed from the

axotomized condition, in agreement with Kuno et al. (1974b). This could






-81-


indicate that functional reinnervation is not sufficient for recovery of

motoneuron electrical properties or that time is required for these

parameters to reach levels permitted by reconnection.

At the med-re and long-re stages, motoneurons without functional

reconnection to muscle (non-contracts) have electrical properties

different from those motoneurons which elicit muscle contraction. In

particular, the relationships between rheobase and input resistance, and

between AHP half-decay time and axonal conduction velocity, are

different in these cells. At the long-re stage, all motoneurons with

functional connections had electrical properties similar to normal MG

motoneurons innervating the same type of motor unit. At the med-re

stage, motoneuron properties were not fully recovered, but all cells

which elicited muscular contraction showed electrical properties altered

significantly from axotomy (in the direction of controls), and

segregated according to motor unit type with respect to rheobase, input

resistance, and AHP half-decay time. Collectively, these data suggest

that functional reconnection is necessary for re-establishment of

motoneuron electrical properties. In agreement with our results are

those of Gordon and Stein (1982b) who reported a subpopulation of axons

which did not elicit muscle contraction, and which did not recover

normal action potential amplitude extracellularr; a function of axonal

diameter; see also Gordon, 1983; Gutman and Sanders, 1943). All axons

eliciting muscle contraction in their study recovered to normal levels

of action potential amplitude by nine months to one year

post-operatively.






-82-


Thus, our data suggest that functional reconnection is necessary and

perhaps a sufficient condition for recovery of motoneuron electrical

properties. A similar situation occurs in the goldfish Mauthner cell,

where membrane electrical properties do not recover in the absence of

connection to an end organ (Faber, 1984). In contrast, Kuwada and Wine

(1981) found that the effects of axotomy on somatic excitability of

crayfish central neurons was a transient one, regardless of whether the

axon regenerated and formed new connections. There appears to be

variability in the degree to which the periphery influences neural

properties (Gordon, 1983).

Price (1974) suggested that the motoneuron soma receives two types

of signal from its periphery, an axon-to-soma signal concerning the

state of the axon, and a muscle-to-nerve-to-soma signal, dealing with

the relationship between muscle and nerve. Consistent with this, many

changes seen with axotomy in sympathetic ganglion cells can be induced

by blockage of axonal transport by colchicine (Purves, 1976). Recent

evidence suggests that muscle-derived trophic factors exist (reviewed in

Slack et al. 1983), and that uptake of chemical signals from muscle is

activity-dependent (Watson, 1969; Brown and Ironton, 1977; Duchen and

Strich, 1968; Czeh et al. 1978). Block of action potential conduction

with tetrodotoxin (TTX) has been shown to alter AHP duration in a way

similar to axotomy in cat soleus motoneurons (Czeh et al. 1978), as well

as AHP half-decay time, axonal conduction velocity, rheobase, and input

resistance in cat MG motoneurons (Munson et al. 1985). Electrical

stimulation distal to, but not proximal to the TTX cuff was observed to

reverse the change in AHP duration in soleus motoneurons





-83-


(Czeh et al. 1978). Further experiments (Gallego et al. 1979) suggest

that the metabolic state of the innervated muscle is the important

variable, rather than activity per se. Thus the muscle exerts influence

on the expression of motoneuron electrical properties.

Recognition of motor unit types and recovery of properties.

A second goal was to determine at what stage motor unit types are

first recognizable, and how this relates to the level of recovery of

motoneuron properties. We found that motor unit types became

recognizable using contractile criteria at the med-re stage (8-10

weeks), before mean motoneuron electrical properties and mean muscle

unit contractile properties had reached mature levels.

Although mean values for motoneuron electrical properties were not

at normal levels, motoneurons segregated into types with respect to

rheobase, input resistance, and AHP half-decay time at the med-re stage.

At this stage most axons had made functional connections with muscle

fibers, although apparently some axons destined to make connections had

not yet done so. Relationships between motoneuron properties, between

muscle unit speed and tension, and between motoneuron and contractile

parameters, were relatively normal at the med-re stage.

Most of the increase in muscle and motor unit tension beyond the

med-re stage can be accounted for by increase in muscle fiber area,

although there may be a small increase in number of axons with

functional reconnection. Motor unit tension recovery follows the pattern

of muscle fiber area recovery. Muscle fiber area increased 160% from the

med-re to the long-re stages, whole muscle twitch tension increased

120%, and mean motor unit tetanic tension increased 129%. Type FG muscle









fibers did not fully recover cross-sectional area, thus type FF motor

unit tetanic tension was reduced. This failure of recovery could reflect

altered activity (Lapointe and Oardiner, 1984; Spector, 1984), altered

trophic relationships between nerve and muscle (Spector, 1984; Lapointe

and Oardiner, 1984; but see Cangiano and Lutzemberger, 1980), or a

mismatch between motoneuron firing rate and the muscle's ability to

respond (Lowrie and Vrbova, 1984). In general, these data confirm the

suggestions made by Gordon and Stein (1982b) for self-reinnervation of

cat MS.

At the med-re stage a greater than normal proportion of the fast

units were fatigue-resistant, as indicated by the reduced proportion of

type FF, and increased frequency of types FI and FR motor units. It is

likely that some motoneurons, which originally innervated type FF units,

innervated more fatigue-resistant types FI and FR units at this stage.

Lowrie and Vrbova (1984) reported increased fatigue resistance in rat

muscle after crush injury to the nerve. Perhaps the increased resistance

to fatigue reflects increased activation of type FF units due to the

lowered tension of all motor units (see also Baldwin et al. 1984).

Increased fatigue resistance could also reflect increased activation

during the stage of poly-neuronal innervation seen with reinnervation

(McArdle, 1975; Letinsky et al. 1976; Rothshenker and McMahan, 1976), or

be a general feature of the metabolism of differentiating muscle.

Increased fatigue resistance of fast muscle is also a transient

phenomenon in developing rat muscle (Lowrie and *-bova, 1984). By nine

months, fatigue resistance of self-regenerated cat MG units was as in

normal MG (CHAPTER III; Chan et al. 1982; Burke, 1980).






-85-


There were no correlations between muscle tension and speed, axonal

conduction velocity and twitch time-to-peak, or axonal conduction

velocity and muscle tension at the low-re stage, but all these

relationships had recovered by the med-re stage, when mean whole muscle

tension was about 60% of that at nine months self-reinnervation. Gordon

and Stein (1982b) reported that the normal relationship between unit

contractile speed and twitch tension was lost in newly reinnervated MG,

but recovered when whole muscle tension was about 50% of that eventually

attained (3 months post-operative). In their study the relationship

between tension and axonal action potential amplitude recovered more

slowly (six months).

Gordon and Stein (1982b) suggested that the initial lack of

correlations was due to the early-reinnervated units being heterogeneous

in nature, since axons do not return to their original muscle fibers

(Karpati and Engel, 1968; Kugleberg at al. 1970). In support of this

notion, we find that recovery of relationships between motoneuron and

muscle properties is coincident with the re-establishment of recognizable

motor unit types. Also, muscle fiber type grouping was first evident at

the med-re stage, suggesting that motoneurons had induced myosin and

mitochondrial enzyme properties of their muscle unit by that time.

Motoneuron properties and muscle unit properties were re-established

with a similar time course.

Relative reinnervation of motor unit types. A third question was

whether any motor unit type might be favored in the process of

self-reinnervation of a mixed muscle. From the earliest stage that motor

unit types could be recognized (med-re), there was no difference in the






-86-


relative proportions of fast (FF+FI+FR) vs. slow (S) motor units,

suggesting no advantage for survival of particular motoneuron types.

We found a slight increase in the proportion of type SO muscle

fibers at the med-re stage. Together with the unchanged proportion of

type S motor units, this leads to an estimate of increased mean

innervation ratio for type S units. This appeared to be at the expense

of type FR units (type FOG muscle fibers). We observed a similar

situation after nine months self-reinnervation (CHAPTER III). Since

motor unit types could not be recognized before the med-re stage, it is

not possible to determine whether this slight advantage was due to

different rates of axonal growth (e.g. type S axons reaching the muscle

first) or more successful competition after contacts were made. These

data could be evidence for a slight advantage of type S motoneurons in

competition for muscle fibers during reinnervation as suggested by Lewis

et al. (1982; see also Ip and Vrbova, 1984).

Thus there is no strong evidence for an advantage in reinnervation

by any motor unit type, although type S units may be more effective in

competing for muscle fibers (see also CHAPTER III).

Comparison with ontoRenv. There were some parallels between the

recovery of motor unit properties following self-reinnervation, and the

progression of normal ontogeny. The above mentioned transient phase of

high fatigue resistance was one similarity (Lowrie and Vrbova, 1984).

The resemblance between axotomized adult motoneurons and motoneurons of

young kittens with respect to action potential overshoot, AHP duration,

and axonal conduction velocity, as well as the relationship between AHP

duration and conduction velocity led Huiszar et al. (1975) to suggest






-87-


that axotomy results in a dedifferentiation of motoneuron properties to

a level similar to early development.

Most motoneuron and motor unit properties that have been

investigated in kittens differentiate to the adult pattern by the sixth

to tenth post-natal weeks (Kellerth et al. 1971; Bagust et al. 1973;

Hammarberg and Kellerth, 1971; Hammarberg, 1974; Nystrom, 1968). This

corresponds to the time at which kittens acquire the ability to run, and

to the time of weaning (5-6 weeks; Peters, 1984).

During ontogeny, motoneuron soma size (Sato et al. 1977; Mellstrom

and Skoglund, 1969), dendritic morphology (Scheibel and Scheibel, 1970),

and axon myelinization (Berthold et al 1983) continue to mature well

beyond this 6-10 week period. Relative to these morphological changes,

axonal conduction velocity does not reach adult values until late in

development (although fast vs slow axons are differentiated from birth,

or close to that time; Ridge, 1967; Huiszar et al. 1975; Bagust et al.

1973). Also, although the difference between AHP duration of fast and

slow motoneurons is established by ten weeks, adult values are not

attained until late in development (Hammarberg and Kellerth, 1975),

perhaps related to the increase in dendritic tree development. This is

especially true for the slow, soleus motoneurons (Euiszar et al. 1975).

Following axotomy, motoneuron properties dedifferentiate. If

reinnervation of muscle is allowed, motor unit types are again

recognizable by 8-10 weeks. Motoneuron and muscle unit properties mature

in parallel, not reaching control levels until long after motor unit

types are recognizable. The pattern of recovery of motoneuron electrical

properties is shown in Fig. 4-5.







-88-


- C 0r -S


C.


--- --4


WI -.C S-t


a1 ------.---- -----------*
-* *u o t -> -S( i'rt- t
ril cw St *.-i*-'t.,










%,
I \
j ~ ^ '


-t ftt WI) it
*~Y )t li'~*r Y() C


-_4


>*0 4 -


' SC i^ t -, **-C ON,:-Wf
t...( .t *St*M.E*t i.


Figure 4-5. The time course of recovery of overall means for motoneuron
electrical properties. (A) Rheobase decreases with axotomy, then
gradulaly recovers to normal levels with time after reinnervation. (B)
Input resistance increases with axotomy, then gradually recovers to
normal levels with time after reinnervation. (C) AHP half-decay time.
The mean values do not change with axotomy of reinnervation. (D) Axonal
conduction velocity decreases with axotomy, then gradually recovers to
control levels by nine-months post-operative.


- ------------


A I'

r


- ----~









Huiszar et al. (1975) found no clear relationship between

alteration of particular motoneuron properties and changes in whole

muscle twitch parameters, during postnatal development in oat MG and

soleus. Similarly, we found no clear relationship between alteration of

single motoneuron properties and changes in muscle unit properties

during self-reinnervation of MG in adult cats. Rather, the overall

motoneuron character (as indicated by motoneuron type) was strongly

related to muscle unit type.

While there were many parallels with ontogeny, self-reinnervation of

adult muscle is not a strict recapitulation of events occurring in

development. In regeneration there are additional connective tissue

barriers to axonal growth, and cues for axonal guidance are likely to be

different (reviewed in Gordon, 1983; Vrbova et al. 1978). For example

the basal lamina adjacent to the former endplate region is known to

attract regenerating axons (Sanes, 1983). In regenerating adult muscles

all muscle fibers are present before axonal ingrowth, and these muscle

fibers have already reached a state of differentiation which was lost to

an uncertain degree following loss of innervation. Although the results

of reinnervation studies such as this one suggest that muscle fibers

possess considerable plasticity, it is not certain whether this ability

to alter properties is complete. The increased proportion of type SO

muscle fibers following reinnervation could represent a type difference

in muscle fiber or motoneuron plasticity.















CHAPTER V
PROPERTIES OF NORMAL LG AND SOLEUS

Introduction



The cat lateral gastrocnemius (LG) muscle has been an important

model in studies of anatomical and histochemical compartmentation of

muscle (English and Letbetter, 1982a,b; English and Weeks, 1984; Weeks

and English, 1983, 1985), sensory partitioning within muscle (Van den

Noven et al. 1983), differential usage of muscle compartments (English,

1984; Russel et al.1984; Rushmer et al. 1984), and motor reinnervation

of muscle (Gordon and Stein, 1982a). Despite its importance, there are no

detailed reports of motor unit contractile properties or distribution of

motor unit types in this muscle, and even less information about

motoneuron electrical properties. A few LG units are included in studies

by Burke (1967; 14 units), Burke et al.(1973; 12 units), Burke et al.

(1982; 12 units), Pinter et al. (1983, unknown number of units) and

Hammarberg and Kellerth (1974; 36 units). These motor units appeared

similar to those in medial gastrocnemius (MG), and were combined with

data for that muscle in these reports. Gordon and Stein (1982a) present

data for motor unit type distribution and unit tetanic tension from LG

and soleus units combined. Hammant (1977) studied axonal conduction

velocity and contractile properties for 76 LG units, and suggested that

LG may differ from MG in possesing a higher proportion of fatiguable

units, fewer type S units, and smaller tetanic tensions.


-90-