The Transduction properties of intercostal muscle afferents

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Title:
The Transduction properties of intercostal muscle afferents
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iv, 143 leaves : ill. ; 29 cm.
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Holt, Gregory Alan
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Subjects / Keywords:
Intercostal Muscles -- physiology   ( mesh )
Intercostal Muscles -- innervation   ( mesh )
Mechanoreceptors -- physiology   ( mesh )
Afferent Pathways -- physiology   ( mesh )
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bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1992.
Bibliography:
Includes bibliographical references (leaves 135-141).
Statement of Responsibility:
by Gregory Alan Holt.
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Typescript.
General Note:
Vita.

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University of Florida
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THE TRANSDUCTION PROPERTIES OF
INTERCOSTAL MUSCLE AFFERENTS



















By

GREGORY ALAN HOLT


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


1992














TABLE OF CONTENTS



ABSTRACT . . iii

CHAPTER 1 GENERAL INTRODUCTION . 1

CHAPTER 2 LITERATURE REVIEW . 3

The Intercostal Muscles .. .. 3
The Muscle Spindle . .. 10
The Golgi Tendon Organ . .. 30

CHAPTER 3 METHODS . .. .. 38

Surgical Preparation ..... . .. 38
Displacement Protocol .. 45
Velocity Protocol . .. .. 50
Analysis . . 57

CHAPTER 4 RESULTS . . 59

Muscle Spindles . . 59
Displacement Coding 10 Muscle Spindles 62
Velocity Coding 10 Muscle
Spindles . .... 70
Displacement Coding.-.2. Muscle Spindes 80
Velocity Coding 20 Muscle
Spindles .. . 85
Golgi Tendon Organs . .. 97
Displacement Coding Golgi tendon
organs .. .. .. .... . 97
Velocity Coding Golgi tendon
organs .. . .. 97

CHAPTER 5 DISCUSSION .. . 108

REFERENCE LIST. . . 135

BIOGRAPHICAL SKETCH ...... .. . 142














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

THE TRANSDUCTION PROPERTIES OF
INTERCOSTAL MUSCLE AFFERENTS

By

GREGORY ALAN HOLT

August 1992


Chairperson: Paul W. Davenport, Ph.D.
Major Department: Physiology

Intercostal muscle afferents sense length and tension

changes in the rib cage and transmit this information to the

central nervous system in the form of a neural code. The

mechanical transduction properties of these muscle afferents

are presently unknown. The purpose of this study was to

characterize the coding of dynamic and static mechanical

changes in the intercostal space (ICS) by these afferents.

These experiments were performed on cats that were

anesthetized, instrumented, and artificially ventilated.

Fifty-six afferents were localized with the majority being

found in the more dorsal intercostal region. Eleven primary

muscle spindles, ten secondary muscle spindles, and ten Golgi

tendon organs were localized within the isolated 7th

intercostal space by single unit dorsal root recordings. The

transduction properties of these afferents were determined by


iii








iv

mechanically controlled displacement and velocity changes

within the intercostal space that encompassed the physiologic

range of the muscle. The peak instantaneous, mean ramp,

static discharge, and static hold phase frequencies were

recorded to characterize dynamic coding of intercostal muscle

mechanics. Displacement coding was characterized by the

static discharge frequencies. Primary afferents were found to

possess velocity and displacement sensitivities of 3.0 spikes'

sc/m'm-m", and 8.8 spikes/sec/mm, respectively. Secondary

muscle spindles were found to possess velocity and

displacement sensitivities of 0.6 spikes'e"/m'"", and 3.7

spikes/sec/mm, respectively. Golgi tendon organs were found

to possess velocity and displacement sensitivities of 2.4

spikes'se-/m'-"e, and 2.7 spikes/sec/mm, respectively. It was

determined from these studies that

I. Intercostal muscles possess respiratory muscle

afferents that transduce muscle mechanics into a definable

neural code. These studies indicate that these receptors are

sensitive to stretch within a physiologic range.

II. Primary muscle spindles have a greater dynamic and

static response than secondary muscle spindles.

III. The Golgi tendon organ's studied are found to be

responsive to the dynamic and static phase of stretch, but did

not possess coding sensitivities greater than the range of the

muscle spindles tested.














CHAPTER 1
GENERAL INTRODUCTION



The intercostal muscles of the mammalian thorax have been

shown to be richly innervated by sensory receptors. (Critchlow

& Von Euler, 1963; Von Euler & Peretti, 1966; and Duron et

al., 1978). These respiratory muscle afferents have also been

demonstrated by a number of authors to respond to changes in

both respiratory muscle length and tension. This sensitivity

can be described by the static and dynamic firing frequency of

these respiratory mechanoreceptors that supply the CNS with

respiratory muscle proprioceptive information in the form of

a neural code.

The neural code transduced by intercostal muscle

afferents result from length and tension changes in the

intercostal muscle. This information is received in central

neural sites and serves to modify respiratory neural drive to

optimize respiratory muscle pumping. While it is known that

these mechanoreceptors are responsive to muscle mechanics, the

specific afferent response to controlled changes in muscle

mechanics is unknown. Therefore, it is important to determine

the response of these mechanoreceptors to changes in

respiratory muscle mechanics to understand the role of these

afferents in the control of respiration. The specific








2

hypothesis of this project is that intercostal muscle

afferents transduce respiratory muscle mechanics into a

definable neural code.














CHAPTER 2
LITERATURE REVIEW



The Intercostal Muscles


Anatomically, the human external intercostal muscles

extend posteriorly from the tubercle of each rib anteriorly

toward the costo-chondral junction where they become

continuous with the anterior intercostal membrane. These

muscles extend from the caudal border of the cranial rib and

insert on the cranial border of the caudal rib with their

fibers sweeping down obliquely and ventrally. The internal

intercostal muscles extend anteriorly from the sternum to the

angle of the ribs where the fibers become continuous with the

thin posterior intercostal membrane. These fibers originate

from the inner ridge of the caudal border of each cranial rib

and corresponding costal cartilage and insert on the cranial

border of each caudal rib. The fibers of the internal

intercostal muscles sweep downward and posteriorly in each rib

space. The external and internal intercostal muscles exist as

eleven pairs and are innervated by the external and internal

intercostal nerves (Gray, 1974).








4

Historically there has been much debate about the

function of the intercostal muscle. The theory that has

gained general acceptance has been attributed since the late

eighteenth century to the physiologist Hamberger (Duron,

1973). He suggested that the external intercostal muscles are

activated during inspiration and function to raise the ribs,

thereby increasing the anterior-posterior (A-P) diameter of

the chest (Hulliger, 1981). The internal intercostal muscles

are thought to be stimulated during active expiration. This

theory has been supported by evidence from electromyographic

(EMG) recordings in cats (Greer & Stein, 1989), dogs (Farkas

& DeTroyer, 1987), and humans (Green & Howell, 1959; Taylor,

1960; and DeTroyer et al., 1983). Support has also been found

with efferent recordings of isolated intercostal nerves

(Sears, 1964b), and studies of rib cage muscle mechanics

(Wade, 1954; Goldman & Mead, 1973; and DeTroyer & Sampson,

1983). Campbell et al. (1955), and subsequently Green and

Howell (1959) and Taylor (1960), described the responses of

the human intercostal muscles during respiration. Campbell

found that EMG activity from the 7th intercostal space (ICS)

appeared to be increased with an expiratory effort > 40

cm/H20. At an inspiratory effort of the same magnitude there

was a similar response found at the ICS while the external

oblique became silent. This suggests that the primary

function of the external intercostals is inspiratory, and the

reason for the activity during an expiratory effort could be








5

due to signals from other expiratory muscles, including the

internal intercostals. Green and Howell (1959) also found the

EMG activity of the external intercostal muscles to continue

into early expiration suggesting an inspiratory function

resulting from a decrement in activity towards the end of

maximal airflow. Additionally, in a review of respiratory

mechanics, Otis, Rahn, and Fenn (1950) concluded the

intercostal muscles alone could maintain adequate pulmonary

ventilation. This conclusion was based in part on a study of

older German literature that provided an approximation of

intercostal tissue mass. Assuming that there is a comparable

maximal level of activation and force produced between

intercostal muscles and other muscle groups, it was calculated

that the work that could be produced by the intercostal

muscles would lie approximately between 8 and 13.5 kg'm. This

value would predict the area of lung function in a pressure-

volume diagram, and, as expected, the area calculated from one

study was 8.7 kg'm (Fenn, 1951). Otis later provided evidence

in support of the function of the internal intercostal muscles

in an elaborate paper on the work of breathing (Otis, 1964).

Here, Otis describes detailed mathematical evidence, based on

tissue mechanics and the pressure-volume diagram, for

sufficient potential energy storage at the termination of a

normal tidal breath for exhalation to be a passive event.

This is supported by both physiologic and anatomic studies,

specifically electromyographic evidence and observations of








6
the decreased thickness of the internal, compared to the

external, intercostal muscle. The importance of the

intercostal muscles in respiratory function was further

demonstrated by D'Silva et al. (1953) who found a 20 30%

reduction in maximal ventilation in patients with the

restrictive disorder, ankylosing spondylitis. This condition

results from an inflammation and fusing of the vertebral

joints causing rigidity in the spinal column and thorax. From

these experiments, and also in a later study by Zechman et al.

(1977), it was found that in subjects fitted with a

restrictive rib cage jacket, a similar decrease in vital

capacity (30%) was induced. In comparison, Green et al.

(1978) found a 5% reduction in vital capacity with little

change in tidal volume (92% of control) in subjects fitted

with a restrictive jacket due to an increase in both diaphragm

activation (an average increase of 55%) and abdominal

displacement. These results were also supported by findings of

Konno and Mead (1967) who used A-P measurements of the chest

wall and of the abdomen during isovolume maneuvers in man.

They found that with the rib cage in a fixed position, changes

in abdominal circumference could account for 68% of the lung

volume change during eupneic breathing in a supine posture.

During a vital capacity maneuver they concluded that rib cage

circumferential changes could account for up to 70% of the

total vital capacity. Results from other researchers also








7
demonstrate that the A-P diameter of the chest wall is

significantly reduced by intercostal muscle paralysis (Polgar,

1949; and DaSilva et al., 1977).

The mechanics of chest wall movement and the activation

of the intercostal muscles are still the subject of

controversy, however. Duchenne (Campbell, 1955) was the first

to describe in the 1860s the fulcrum type of effect that the

abdominal contents impose on the thoracic cage as the

diaphragm descends. Duchenne showed an increase in rib cage

diameter with inspiration in the intact animal and thoracic

contraction in the eviscerated animal. In more recent

studies, other researchers (DeTroyer & Kelly, 1982; and

DeTroyer et al., 1983) have shown both the internal and

external intercostal muscles of dogs to be activated during an

inspiratory maneuver from functional residual capacity (FRC).

Additionally, the studies of Wade (1954) have shown that the

movements of the rib cage during inspiration varied widely

between subjects that have adapted to different breathing

strategies. Primrose (1952) considered the intercostal

muscles to play a small role during eupneic breathing, but

found from an accidental observation that intercostal muscle

activity was greatly enhanced when the animal (rabbit) was

made to respire through a resistive airway. He furthered this

suggestion of intercostal muscle function from observations of

asthmatics during an acute episodic attack (Primrose, 1952).








8
The changes in length of the intercostal muscles during

respiration have also been studied using several different

techniques. In a study of intercostal afferents, Von Euler et

al. (1966) observed that during normal spontaneous ventilation

in cats, intercostal muscles had length excursions of

approximately 2 mm. Discreet changes in intercostal rib space

dimensions were later measured during spontaneous breathing

using sonomicrometry. Greer and Stein (1989) showed the

greatest amount of shortening to be present in the more

rostral spaces compared to the mid-thoracic spaces (1.6-9.2%

and 0.7-3.3% of muscle length measured at FRC, respectively).

They also described a shortening of the most caudal spaces

during inspiration that was not related to intercostal muscle

activation, but due to diaphragmatic/rib-cage interactions.

Roentgenographic studies of lateral chest wall movement

by Polgar (1949) introduce the concept that the elevator costae

muscles provide the force for primary chest wall expansion.

From examination of the skeletal attachments of the elevator

costalis muscles between the vertebral transverse processes

and the dorsal surface of the bony rib, it is observed that

they are responsible for considerable abduction of the rib.

This actively increases the lateral chest wall diameter while

rostral movement in the horizontal plane is controlled by

actions on the intercostal muscles and from passive actions on

the rib cage by the diaphragmatic contraction on the abdominal

contents. A recent study by Goldman et al. (1985) extended








9
these findings using human EMG recordings of elevator costae

muscles at the spinal level of T9 and T10. They found

rhythmic inspiratory activation that they concluded to be an

aid in lower rib cage expansion due to a decrease in the

mechanical interactions with the sternum and adjacent rib

spaces. The suggestion that the elevator costae muscles may be

primary for chest wall expansion has been met with some

difficulty, however, owing to the fact that human elevator

costae are greatly reduced in functional size compared to

reptilian elevator costae that provided the initial basis for

Polgar's argument.

The primary conclusion from the literature is that any

force that produces the movement of the rib in either a

vertical or a horizontal plane will cause an increase in the

A-P diameter of the chest wall. It is by the normal excursion

of the thoracic cavity through its range of motion that allows

for the range of respiration to be accounted for at a minimal

cost of breathing.

More importantly, in reference to this project, it has

been demonstrated by Von Euler and other researchers

(Critchlow & Von Euler, 1963; Sears, 1964; and Duron et al.,

1978) that the extrafusal fibers of the external and internal

intercostal muscles have a number of sensory end-organs.

Other authors (Holmes & Torrance, 1959; Godwin-Austen, 1969;

Shannon, 1986; Halata, 1988; and Holt et al., 1991) have also

shown the joint capsule of the posterior vertebral-chondral








10

junction to be innervated by specialized joint

mechanoreceptors. Together, these respiratory related

mechanoreceptors, acting in a similar fashion to those

afferents in other muscle groups, transduce intercostal muscle

proprioception and mechanics into a neural code that is

conveyed to the central nervous system for integration at the

level of the spinal cord (Aminoff & Sears, 1971; Remmers,

1973; and Smolin & Frankstein, 1979) and higher brain centers

(Decima, 1969; Shannon, 1986; Gandevia & Macefield, 1989; and

Davenport et al., 1991).


The Muscle Spindle


Historically, the discovery of the muscle spindle is

attributed to Kolliker and Kuhn (Hulliger, 1984) in the late

1860s. They found the muscle spindle to be fusiform in shape

and comprised of both muscle and nervous tissues. Debate over

the purpose of these muscular elements continued for next 30

years. Interestingly, it was Kuhn that chose the term muscle

spindle that described what was known, i.e. morphologic

appearance. Sherrington (Matthews, 1963) resolved the issue

of muscle spindle function in 1864. Sherrington transected

ventral roots and found that the extrafusal fiber atrophied

while the muscle spindles with their afferent projections were

left intact. Sherrington hypothesized that muscle spindle

morphology and their nervous system connections were not

compromised by the loss of efferent drive. This would








11

indicate that the physiologic function of muscle spindles

would also be left intact, and that this function would be

sensory in nature. In the next four years, Ruffini (Matthews,

1964) provided the first drawings of the isolated muscle

spindle using gold impregnation staining techniques. His

illustrations provided evidence for the fusiform nature of the

muscle spindle and the connective tissue capsule surrounding

the spindle. More importantly, he described the primary and

secondary endings of the afferent nerves on the nuclear bag

and chain intrafusal muscle fibers and the motor end plate

region that is supplied by the gamma motoneurons. From these

early and subsequent morphologic studies, it became apparent

that muscle spindles existed in a variable range of fiber

arrangement and proportions. The muscle spindle itself can be

seen to contain 8 to 10 intrafusal neural elements with 2 5

being primary afferents contained within a connective tissue

capsule. Today it is generally accepted that the primary

afferents are either Group I (between 12 and 20 pm in

diameter) or Group II (between 4 and 12 Am).

Recordings of multiple muscle spindle afferents were

initially made by Adrian and Zotterman (Hulliger, 1984) in

1926. It was later, in 1931, that the first recording of a

single isolated muscle spindle afferent was made by B.H.C.

Matthews (Matthews, 1963). It was the classical studies by

B.H.C. Matthews in frog and cat muscle that first led to a

description of the type of information being transduced by








12

these receptors. He found these to be rapidly adapting to the

dynamic phase of muscle stretch and slowly adapting to the

static phase. The dynamic phase of muscle stretch is defined

as the active phase of muscle fiber length change. The static

phase of muscle stretch is defined as the inactive phase of

muscle fiber length change, i.e., a nonmoving state. Matthews

also found that there could be two effects produced by

muscular contraction: 1) contraction of the muscle mass

surrounding the spindle would cause the spindle discharge to

be transiently terminated due to the parallel arrangement of

the spindle in relation to extrafusal fibers, or 2) an

increase in the contraction stimulus would cause the spindle

to be activated, which he originally suggested to be due to

activation of smaller diameter efferent fibers causing

contraction of the muscle spindle (Kuffler et al., 1984).

These experiments gave support to a general hypothesis that

muscle spindles were responsible for the transduction and

conveyance of muscle length and position sense to the CNS.

Leskell (Hulliger, 1984) provided evidence for the

fusimotor control of muscle spindles in 1945. In his

experiments he applied a pressure block on the larger alpha-

extrafusal fibers supplying a specific muscular region and

then stimulated the corresponding ventral root. In this

manner he was able to stimulate both alpha and gamma motor

nerve fibers but only the smaller gamma fibers supplying the

intrafusal muscle fibers would pass the block. He observed








13

that stimulation with the block elicited no significant change

in muscular tension, but a significant increase in multi-fiber

afferent discharge was recorded. Hunt and Kuffler (1951a & b)

then used two single-unit preparations connected to both a

gamma-efferent nerve and an afferent fiber supplying a single

muscle spindle. By stimulation of the gamma efferent fiber

from a ventral rootlet they observed an increased afferent

discharge from the muscle spindle with no appreciable change

in tension in the surrounding muscle. These experiments

demonstrate the ability of the muscle spindle to contract in

the same manner as the surrounding extrafusal fibers. Thus,

it was shown that the sensitivities of this type of

mechanoreceptor could be adjusted to the velocity and overall

contractile length of the inhabited muscle.

However, physiological and anatomical questions remained.

The primary and secondary intrafusal fiber types originally

described in the spindle have subsequently been termed nuclear

bag and nuclear chain fibers, respectively. This terminology

is the result of the recognition of nuclear arrangement in the

intrafusal fiber. P.B.C. Matthews (1964) described two types

of gamma-efferent motoneurons innervating the muscle spindle.

The gamma-static motoneurons supplying the chain fibers in the

trail type ending, and the gamma-dynamic motoneurons

innervating the bag fibers in the plate type ending (Figure 2-

1). The plate type of gamma-dynamic efferent ending refers to













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16

a more localized area of innervation than that they described

for gamma-static efferent innervation. Both nuclear bag and

chain fibers were shown to be innervated by gamma-dynamic

afferent fibers at their equatorial regions. The innervation

of these gamma-dynamic afferent fibers had also been termed

annulospiral endings. This was evidenced by the morphologic

appearance of the afferent endings that showed a spiral

wrapping of the equatorial region of the intrafusal fiber by

the afferent fiber. Additionally, Matthews described a

preferential innervation of gamma-dynamic afferents to nuclear

bag fibers. Similarly, the gamma-static afferents innervate

both types of intrafusal fibers at their polar regions with a

preference for the nuclear chain intrafusal fibers. This type

of afferent fiber termination has also been termed flower

spray ending, for the morphological appearance of the sensory

terminal on the intrafusal fibers. The validity of this

schema was found to contain certain exceptions as suggested by

Barker et al. (1973) who considered the innervation by the

gamma system to be non-specific. He described two types of

static efferents that could be found to end as trail endings

on both nuclear bag and chain fibers. It later became

apparent that there are indeed at least two types of bag

fibers, designated bag, and bag2. Morphologically, the bag2

fibers more closely resembled the classical nuclear bag fiber

but was innervated by a static gamma-motoneuron. Confusion as








17

to the specificity of gamma innervation rapidly developed with

one consistency being that a single intrafusal fiber is

innervated by a single gamma efferent fiber.

The functional significance of the two types of

afferents, primary and secondary, was demonstrated by the

studies of Cooper (1961). This researcher isolated muscle

spindles in decerebrate cats and differentiated between

afferents of primary and secondary endings by conduction

velocity testing. It had been shown by earlier authors that

the conduction velocity of the primaries is considerably

faster than that of the secondaries (70 120 M/sec and 30 -

70 M/sec, respectively). Cooper found that the secondary

afferents would discharge with the same frequency as the

spindle primaries at a constant muscle length, but did not

possess the dynamic properties during muscle stretch as did

the spindle primaries. It has eventually become accepted that

the afferents terminating on the bag2 fibers transduce muscle

length information concerning muscle length. The afferents on

nuclear bag, fibers transduce acceleration and velocity of the

muscle stretch. Techniques to differentiate between these two

types of afferents then became necessary due to a large

overlap that was soon found between their conduction

velocities. It was from these preceding studies and

subsequent morphologic evidence that the purpose of this








18

receptor was identified. Morphologically, an increased

density of spindles was found in muscles controlling fine

motor tasks.

The original studies of the functional role of the muscle

spindle in the control of respiration were conducted by Von

Euler and coworkers. Through an elegant series of

experiments, Von Euler was able to show that many of the

muscle spindles in the intercostal muscles of the rib cage

function with a high level of gamma-efferent activity.

Critchlow and Von Euler (1963) recorded from 168 muscle

spindle afferents in 41 cats. Using single-fiber dorsal root

recordings that corresponded to intercostal spaces 5 8,

these authors found that during tracheal occlusion, there was

an initial significant increase in spindle activity followed

by a marked decrease. The events being signalled are an

immediate increase in respiratory muscle tension and a

concomitant decrease in muscle length. The authors considered

this to be due to dual activation of the gamma/alpha efferent

system that was followed by spindle unloading by the

extrafusal fibers or by a spinal inhibition of the gamma

drive. Many of the afferents that were found to be

inspiratory active were under considerable gamma control as

shown by weak anaesthetic (0.25% lidocaine) application to the

intercostal nerve. In the control group of spindles, the

highest frequency of discharge occurred during the beginning

and termination of inspiration. There was a loss of the








19

inspiratory activation of the intercostal muscles and a

discharge that occurred only during expiration post lidocaine

administration. This concentration was found by these

researchers to block conduction in the smaller diameter gamma

fibers while leaving the alpha fibers functional. This would

argue that there is a loss of the gamma/alpha activation that

occurs during inspiration and would account for the increased

activity with intercostal muscle shortening. An absence of a

gamma-bias suggests that the mechanoreceptor functions as a

passive length sensor. This is evidenced by afferent

frequency discharge with muscle fiber lengthening that occurs

in expiration. Of the total number of spindles shown to be

under rhythmic gamma control, 22 were found to be inspiratory

active and 9 were found to be expiratory active. The

differentiation was characterized through observations of the

passive properties of the muscle spindles studied.

These results were later supported by Greer and Stein

(1989) who performed studies on gamma-motoneuron activity in

external intercostal muscles of cats. They found tonic gamma-

motoneuron activity present throughout the external

intercostal muscles of the rib cage while rhythmic gamma-

motoneuron activity was found only in the regions that were

active during the respiratory cycle. Those areas that they

had characterized as being active during respiration were from

the 1st to the 7th intercostal space extending dorso-ventrally

for the first 3 spaces and then receding dorsally moving








20

towards the 7th intercostal space. Similarly, in a study of

160 muscle spindles in the intercostal muscles of cats, Von

Euler and Peretti (1966) found 75% to lie between the bony

shafts of the ribs. Twenty percent were located between the

supra-angular portions of the rib space and only 5% were

located between the costal cartilaginous regions. They also

described a decrease in the number of mechanoreceptors found

moving from dorsal to ventral areas.

Another property of spindle function in respiration

suggested by Critchlow and Von Euler (1963) was that of

reflex-induced activation of muscle spindles by other chest

wall proprioceptors. In spontaneously breathing vagotomized

cats, 10 muscle spindles were studied and showed no decrease

in rhythmic inspiratory discharge when artificially over-

ventilated to terminate the spontaneous ventilatory movements.

Nine of these spindles were then shown to exhibit a phase

reversal in discharge patterns following lidocaine injections,

i.e., an increase in frequency discharge with inspiration

became a decrease in frequency discharge with inspiration.

This was considered to be due to the removal of the gamma

drive that was present with and without extrafusal fiber

excitation. Similarly, following high spinal cord

transactions (C8 Tl) Crithchlow and Von Euler (1963) found

that 4 of 5 spindles studied continued to exhibit rhythmic

gamma-bias. Conversely, in a later study Corda et al. (1966)

found a loss of rhythmic gamma motor activity following high








21

cervical spinal cord transaction while tonic gamma motor

activity remained intact. This particular study was supported

by Eklund et al. (1963) who found in spinally transected cats,

muscle spindles in one intercostal space had static activity

that could be affected by either mechanical manipulation or

ventral root stimulation of an adjacent segment indicating a

segmental spinal reflex system. Further evidence for the

reflex control of the gamma-motoneuron system subserving the

intercostal muscles was provided by Corda et al. (1966) using

a decerebrate cat preparation. He found the gamma efferents

to show a greater acceleration of activity following ablation

or cooling of the anterior lobe of the cerebellum. These

researchers also described a significant increase in

ventilation concomitant with the acceleration of gamma

efferent drive suggesting a release of inhibitory influences

on respiration.

Characterization of primary and secondary afferent

response to stretch had been described by a number of

researchers. P.B.C. Matthews (1981) used the soleus muscle of

cats to determine the frequency discharge response to

controlled velocities of stretch to a displacement of 5 6

mm. Matthews recorded from de-efferented primary and

secondary spindles at velocities of 1.2, 5, 15, 30, 50, 75,

and 100 mm/sec. He found both secondary and primary muscle

spindle afferents to possess a dynamic and static response to

stretch, but with characteristic differences between the two.








22

Secondaries increase their discharge almost linearly with the

dynamic phase of stretch. The maximal discharge frequencies

during this phase are dependent on the velocity used, i.e.,

higher velocities result in higher discharge frequencies.

This high discharge frequency is abruptly terminated at the

end of the dynamic phase of muscle stretch and drops towards

static levels. The degree of frequency decrement is also

velocity dependent. In addition, with velocities over 5

mm/sec there is an initial burst present that does not exceed

the maximal discharge frequency. Matthews also demonstrated

that the static response of these afferents is not velocity

dependent and is rapidly attained (< 2.0 sec) following

termination of stretch. In contrast, primary endings had a

characteristic high initial burst response at velocities > 5

mm/sec and linear increases in discharge during the dynamic

phase up to 30 mm/sec and a progressive decrease in discharge

rate at higher velocities. These afferents also had an abrupt

decrease in discharge frequency at the termination of stretch

that was velocity dependent, but the decline towards a static

level was much slower than was exhibited for secondaries.

Importantly, Matthews showed that the static frequency

discharge rates were not markedly different between spindle

primaries and secondaries. The discharge frequencies attained

in this study were found to be approximately 60 spikes/sec for

the static response of both primaries and secondaries while

the dynamic response of primaries was always higher (> 300








23

spikes/sec in some cases) than 20 spindles (> 150 spikes/sec)

and dependent on the velocity of stretch (Matthews, 1981).

Harvey et al. (1961) reported on the response of 55 muscle

spindles (35 primaries and 20 secondaries) to slow stretch in

the soleus muscle of cats. They used displacements up to 12

mm at velocities of 2.3 3.0 mm/sec and found slopes of

frequency discharge to be 8.2 spikes/sec/mm for primary

afferents and 4.3 spikes/sec/mm for secondaries. They found

similar static responses between the two, but the response of

spindle primaries to ventral root stimulation was greatly

exaggerated compared to secondaries.

These researchers also found the spindle secondaries to

have a comparatively higher threshold to stretch of about 1 mm

or 50 g. There are few studies on the response of intercostal

muscle spindles to stretch. Those that have been performed

have used both in vivo and in vitro methods. Von Euler and

Peretti (1966) showed an increase in spindle discharge in a

spontaneously breathing cat with an ICS displacement of

approximately 1.8 mm and a tidal volume of approximately 30

ml. These results were not quantitated, but were calculated

to be approximately 60 spikes/sec during an active inspiration

decreasing to approximately 40 spikes/sec on relaxation.

Newsom-Davis (1975) provided similar results using an in vitro

preparation of excised (approximately 2 cm) human intercostal

muscle. Muscle fiber displacements of 1 mm at velocities of

2 and 6 mm/sec were used and showed the dynamic indices of









spindle primaries and secondaries to be consistent to the

findings of Matthews (1963) and Scott (1990). Newsom-Davis

(1975) also described the effects of a primary afferent to

changes in displacement showing both a higher static and

dynamic frequency discharge with increased length.

Other investigators have reported the characteristics of

spindle discharge in different muscle groups and found them to

be at least partially dependent on the physiologic range of

the host muscle and the surrounding type of extrafusal fibers.

In this respect, one possible hypothesis of a variable

affecting afferent impulse generation came from studies of

extrafusal fiber dynamics in contracting muscles. The

hypothesis suggests that fast contracting extrafusal fibers

can absorb the viscous tension in contracting muscle and thus

decrease the afferent output that would be generated by

dynamic efferent control. Slow contracting extrafusal fibers,

in contrast, would have a higher transient tension and

afferent discharge during muscle contraction due to the lower

crossbridge turnover rate. Therefore it seems possible that

the differences or regional differences in dynamic afferent

discharge characteristics seen in a particular muscle group

may be dependent on the mechanics of the extrafusal fiber type

surrounding the mechanoreceptor.

Differentiation of the muscle afferent type utilizes the

muscle spindle "silent test," first described by Hoffman in

1922 and later by B.H.C. Matthews in 1933 (Higgins and








25

Lieberman, 1968). A positive test indicates a spindle type of

afferent has a transient cessation of discharge in response to

extrafusal fiber contraction due to its parallel arrangement

with the surrounding muscle. This implies that stimulation of

the extrafusal muscle fibers in the region of the receptive

field causes the spindle to become unloaded. The reverse is

true for mechanoreceptors arranged in series with muscle

fibers and these afferents exhibit a negative silent test.

Following a positive muscle spindle silent test, the spindle

afferent can be further classified using a protocol developed

from studies of Matthews (1963), Critchlow and Von Euler

(1963), Jansen and Rudjord (1964), and Scott (1990).

Classification of these muscle spindles will categorize them

as either primary (Group I) or secondary (Group II) spindle

afferents. Afferent nerve fiber conduction velocity testing

has also been routinely used to differentiate between these

two types of afferents. In the cat, primary spindle afferents

have conduction velocities of > 70 M/sec, usually between 70

and 120 M/sec. Secondary spindle afferents have conduction

velocities of < 70 M/sec, usually between 35 and 70 M/sec.

The overlap between the two types of mechanoreceptors is

between 70 and 85 M/sec. Classification of these afferents

using the length of time in the silent period of the spindle

silent test has also been used. These observations suggest

that primary afferents had a shorter silent period (< 75 msec)

compared to secondaries (30 130 msec) (Critchlow & Von








26
Euler, 1963); however, there still was considerable overlap

between the two populations. Later, in a study by Von Euler

and Peretti (1966), it was shown that the Group II secondary

afferents could not discharge in synchrony with muscle

vibration in excess of 400 Hz while some primaries were found

to be able to follow frequencies up to 700 Hz. This technique

was found to possess an error rate of approximately 10%, but

proved to be useful in differentiating between spindles and

Golgi tendon organs. A similar technique was applied by

Bolser et al. (1988) in the selective activation of Ia primary

afferents. Bolser used 100 gm intercostal space displacements

vibrating at 200 Hz and found that Ia afferents could be

stimulated and recorded at the dorsal rootlets. This study

was based on findings of Lundberg and Winsbury (1960) for

selective primary afferent activation at low-threshold

vibratory amplitudes. Cheney and Preston (1976) found that

for primary endings the firing rate at the beginning of the

static hold phase of a stretch was lower than the steady-state

phase, i.e. the firing rate increased to steady-state levels

(discharge frequency taken 2 sec post termination of the ramp

stretch). They also found evidence for secondary afferents

displaying a smooth decrement in discharge frequency to the

static hold phase. Other investigators (Jansen & Matthews,

1962; Lennerstrand & Thoden, 1968; Proske & Stuart, 1985; and

Windhorst et al., 1985) have used the initial burst at the

start of the ramp stretch as another method to characterize








27
spindle afferents. It was later shown that secondary endings

may also have this initial burst characteristic; however,

afferents can be identified as primaries if the initial burst

frequency exceeds the peak firing rate at the end of a 5

mm/sec stretch (Scott, 1989). Burke et al. (1976) performed a

similar study combining afferent frequency discharge

characteristics with ramp stretch and described the responses

of mechanoreceptors in humans. In a series of 23 experiments

on 18 subjects, he recorded the responses of 32 primary

spindle afferents, 13 secondary afferents, and 3 Golgi tendon

organs to a varied frequency of 20 to 220 Hz vibration at an

amplitude of 1500 Am. At this amplitude of vibration they

found that primary afferents could follow vibrational

frequencies up to 220 Hz and while secondary afferents usually

could not be driven past 100 Hz. They also described a

frequency response of Golgi tendon organs to vibration and

showed that these receptors could only respond in a

subharmonic fashion to vibrational frequencies, usually every

second or third cycle at the higher ranges (< 120 Hz). In

addition, these authors described the response of these

mechanoreceptors to the dynamic phase of muscle stretch at a

velocity of 7.50/sec in conjunction with vibration. They

found for primary endings a 1 to 1 cyclic discharge that

ceased with release from stretch. The response of secondary

endings to the dynamic phase of stretch was not as prominent

as that for primary spindle afferents; however, with the








28
release from stretch there was a persistence of afferent

discharge. Scott (1990) reported that to reduce the error

rate in characterization, a series of 5 maximal twitch

contractions from a resting state should be presented to the

surrounding spindle region to provide a stretch history for

the muscle. Additionally, the mechanoreceptor would be

considered primary if there was a clear break in the impulse

discharge at the beginning and end of the of the hold phase of

the ramp stretch. Using these combined criteria in

conjunction with muscle conditioning, Scott found an error

rate of 8% to persist in afferent classification.

A quantitative approach for the characterization of

muscle spindles from the hindlimb muscle of cats was developed

by Matthews (1963). He described the dynamic index (DI) that

could be calculated by taking the difference in the firing

rate at the end of the ramp stretch and the rate 0.5 seconds

after the beginning of the hold phase. In addition, the

difference between the firing rate 0.5 seconds after the start

of the hold phase and the firing rate prior to initiation of

the ramp stretch could be considered as a static index (SI).

Matthews found that primary afferents with conduction

velocities > 70 msec'" showed a high dynamic and low static

index with the reverse being true for secondary afferents.

Similarly, Scott (1990) reported spindle primary values of

41.5 +/- 2.2 spikes/sec S.E. and 31.5 +/- 1.3 spikes/sec S.E.

for dynamic and static indices, respectively. These results








29

also supported the observations of Matthews (1963) for

secondary afferents conducting at < 70 msec'1; the DI was 26.0

+/- 1.4 spikes/sec S.E. and the SI 41.6 +/- 2.06 spikes/sec

S.E.. Combining these two indices into a ratio of DI : SI and

plotting against conduction velocity as the independent

variable, there was a bimodal representation of primary and

secondary spindle afferents. Edin et al. (1990) used the

equation for relative dynamic index = fpak fpateau / plateau -

frest to differentiate between Group I and Group II afferents.

The relative dynamic index for spindle afferents was found to

be 5.5 and 3.0 for 10 and 20 spindles, respectively. This

resulted in a tighter distribution with less overlap of

afferent types. The distribution, however, continued to have

overlap between the two. The final method of afferent

characterization, the dynamic response (DR), was developed by

Jansen and Matthews (1962). The dynamic response is defined

as the difference between the initial burst frequency and

static discharge frequency during the hold phase. Following

muscle conditioning as described previously, Scott (1990)

found the dynamic response for afferents with conduction

velocity > 70 msec"1 to be increased from 44.5 +/- 4.04

spikes/sec S.E. to 83.31 +/- 5.57 spikes/sec S.E. from

observations of pre and post-conditioned states. In addition,

Scott observed that conditioning of the muscle did not affect

the dynamic response of secondary spindle afferents. For

those receptors the DR was found to remain approximately -








30

14.4 spikes/sec. An error rate of 5 10% in spindle

classification is found for each method of classification.

Muscle spindle classification for the experiments in this

project uses a combination of these techniques. These

techniques include the muscle spindle silent test, conduction

velocity, initial burst frequency, and frequency discharge

characteristics over the static discharge and static hold

phase. Classification of receptor position is further

characterized by the rostral-caudal distribution of these

sensory organs within the intercostal muscle space.


The Golgi Tendon Organ


Ruffini (Hulliger, 1981) made the original morphologic

observations of the Golgi tendon organ (GTO) in 1893. These

sensory mechanoreceptors can be described as extensive

branching dendritic trees surrounded and intertwined by the

collagenous fibers of the tendons attaching muscle to bone.

GTOs were described by Skoglund in cat (1956) to exhibit a

varied arrangement and size that is dependent on the

characteristics of the surrounding connective tissue.

Skoglund used gold staining techniques and found branching to

extend up to 1,000 Am and axonal diameter to range from 9 15

im in 20 GTOs observed.

B.H.C. Matthews (Kuffler et al., 1984) initially recorded

tendon organ activity. He found the tendon organ to be

responsive to an increase in the length of the surrounding








31
connective tissue. This response property can be derived from

the morphologic arrangement of the tendon organ in respect to

surrounding tissue, i.e. as the collagen fibers collapse and

simultaneously tighten around the receptor, there is an

increased response displayed as firing frequency. GTO

discharge frequency to static length change was slowly

adapting. Matthews compared the discharge frequencies of

these mechanoreceptors to both length and tension changes

(passive stretch and active contraction of the in series

muscle) and found them to be approximately equal. He

concluded that these afferents are arranged in series with the

muscle. In an later study, Hunt and Kuffler (1951) examined

the effects of stretch and contraction on over 500

mechanoreceptors from the soleus and flexor digitorum longus

of the cat. They found approximately 40% of these to be of

the Golgi tendon organ type. These tendon organs were shown

to possess no steady discharge rate at tensions between 100

and 200 g. Hunt and Kuffler (1951) occasionally found some

GTOs to discharge at 5-10 g tension but generally regarded

them to have a higher threshold for impulse formation compared

to the spindle primaries and secondaries. Jensen and Rudjord

(1964) subsequently found the threshold to discharge for these

Golgi tendon organs to be lower during active contraction than

with passive stretch. Similarly, Reinking et al. (1975)

performed studies on in vitro human GTOs and found no

correlation between increased isolated muscle unit stimulation







32

and firing frequency. They suggested that a better

correlation could be found by taking into account the

mechanical interaction of the receptor and its environment.

Jami and Petit (1976) also found discrepancies in the tensions

produced in different muscle groups and the discharge

responses elicited from tendon organs. The discharge rates

and tensions produced were from isolated motor units during

ventral root stimulation and recorded from dorsal root

ganglia. In recordings from 62 GTOs from various muscle

groups in the cat, they found for example a GTO could reach a

discharge rate of 60 spikes/sec at 11 g tension, but when

tension was increased to 40 g the impulse rate did not exceed

45 spikes/sec. Similarly, a tendon organ that fired between

50 and 100 spikes/sec going from 4.5 to 9.0 g tension, only

showed a discharge rate of 32 to 75 spikes/sec at 14 and 25 g

tension. Houk and Henneman (1967) considered the variable

effects to be due to differences in recruited motor units.

These differences, i.e., numbers of muscle fibers activated

either parallel to or in series with the mechanoreceptor,

create changes in the afferent receptive field and alter its

discharge properties. Passive length changes caused a linear

increase in force acting on these receptors. On the other

hand, the forces acting on the receptor with active

contraction were dependent on the motor units activated.

Should the motor units in series with the receptor be

activated, there will be a decrease in the threshold response








33
with the increased impulse discharges. If, however, the

neighboring extrafusal fibers are stimulated to contract, they

may unload the tendon organ thus bringing it to a subthreshold

level. At a constant length of +5.5 mm (0 being the maximal

physiologic length of the soleus muscle in cat) they found the

discharge pattern to have both dynamic and static properties.

The initial burst (as described for muscle spindles) was

greater than the rise of muscle force and decremented to a

steady-state level. For this particular receptor, the

discharge frequency decreased from an initial burst of 60

spikes/sec to a static level of 48 spikes/sec within 2

seconds. The response to a change in length also displayed

both static and dynamic properties, however, the actual length

change of the muscle or the velocity of the change was not

reported. Houk and Henneman (1967) also found the GTOs to

exhibit a type of saturation effect where the responses to one

motor unit added to that of a second motor unit did not sum

algebraically. Lundberg and Winsbury (1960) found that GTOs

could be separated from Ia afferents by selective stretch

activation. They based their hypothesis on previous studies

by B.H.C. Matthews (Kuffler et al., 1984) and Hunt and Kuffler

(1951a & b) that showed GTOs to possess a higher discharge

threshold than Ia afferents. In a study of 41 tendon organs,

only 6 fired with stretches of < 200 Am while 43 Ia afferents

out of 43 Ia afferents studied discharged at 60 Am or less.

This property of primary afferent activation became of great








34

use in respiration for in vivo studies concerning reflex

effects by selective mechanoreceptor activation (Lundberg &

Windsbury, 1960).

The role of Golgi tendon organs in the intercostal muscle

is yet to be defined. The recent studies concerning the

effects of tendon organ excitation on respiration correspond

with the previously developed hypothesis that GTOs act to

inhibit motoneuron activity to the homonymous muscle to

prevent muscle tearing. Their presence in intercostal muscle

has been studied by Critchlow and Von Euler (1963). They

found that out of 243 afferent fibers recorded, 26 could be

identified as Golgi tendon organs, 168 were found to be muscle

spindles, and 49 were not positively identified. While it is

accepted that they exist in smaller numbers compared to muscle

spindles, later studies concerning their reflex respiratory

function shows that GTOs are capable of inhibiting medullary

inspiratory neurons (Shannon et al., 1987; and Shannon et al.,

1988). The problems that remain to be clarified are the

ranges of GTO discharge and a determination of their role

within a physiologic range. Figure 2-2 is an illustrated

example of a Golgi tendon organ.


Characterization of Golgi Tendon Organs


The basis for GTO identification that will be employed

was developed by B.H.C. Matthews (Kuffler et al., 1984) and by

Jansen and Rudjord (1964). Following receptive field








35

localization by blunt probing, the receptor response to muscle

stimulation will be observed. An augmentation of frequency

discharge is indicative of a GTO response (a negative muscle

spindle silent test).








Figure 2-2. A Golgi tendon organ. The branches of this type of
mechanoreceptor are imbedded in a matrix of connective tissue in
series with intercostal muscle fibers.








37



















/ 4
r- -












CHAPTER 3
METHODS



The focus of this project was to determine the

transduction properties of intercostal muscle

mechanoreceptors. It was hypothesized that intercostal muscle

spindles and Golgi tendon organs transduce muscle displacement

and velocity. The specific aims were:

Aim 1: To show that respiratory mechanoreceptors change

their discharge frequency in response to static changes in

intercostal muscle length and thereby frequency code muscle

displacement.

Aim 2: To show that respiratory mechanoreceptors change

their discharge frequency in response to the rate of change in

intercostal muscle length and thereby frequency code the

velocity of muscle lengthening.


Surgical Preparation


The experiments were performed on cats of either sex

weighing between 2.8 and 3.2 kg. Premedication with atropine

(0.05 mg/kg) was administered to control secretions.

Anesthesia was induced with inhalation of halothane/oxygen

gas. A catheter was placed in the femoral artery to

continuously measure blood pressure (Konigsberg Instruments

No.P36) and obtain arterial blood for gas analysis (Radiometer

38








39
ABL 30). A second catheter was placed in the femoral vein to

deliver fluids and anesthesia. Body temperature was

maintained at 38 +/- 10 by the use of a heating pad (Neco

Model 819) and monitored with a rectal temperature probe (YSI

Instruments). Anesthesia was then switched to i.v. alpha-

chloralose (25 mg/ml). The halothane/oxygen gas mixture was

removed and alpha-chloralose periodically administered to

maintain a deep plane of anesthesia. A tracheal catheter was

also inserted and the animal artificially ventilated (Harvard

Respiratory Pump). Sodium bicarbonate was administered as

necessary for maintenance of arterial pH at 7.4 +/- 0.1.

The animal was then placed in a stereotaxic apparatus and

its head fixed in a nontraumatic head-holder. The spinous

processes T3 and T10 were clamped. A wide skin incision was

made on the left flank and the cutaneous tissues dissected

away to expose the external intercostal muscles. A

laminectomy was performed from T4 to T8 to expose the dorsal

spinal cord. The spinal cord was then flooded with warm

mineral oil and the dura cut longitudinally to expose the

dorsal root filaments. A single rostral dorsal root filament

supplying the 7th intercostal space was severed at its

attachment to the spinal cord and placed on a platform lowered

into mineral oil. The filament was initially placed across

bipolar platinum recording electrodes. The signal was

amplified (Grass P-5), led to an oscilloscope (Tektronix

5111), audio monitor (Grass AM 8) and FM tape recorder (Vetter








40
Model D). The 7th intercostal space was probed to assure that

this intercostal space contained the afferents in the dorsal

root filament.

Initial muscle length (Lii) was measured at functional

residual capacity (FRC) with calipers. The intercostal

muscles of the rostral and caudal rib spaces were severed,

isolating the intercostal space to be studied. Lung inflation

was maintained with a positive end expiratory pressure of 3

cmH20. The rostral rib of the isolated space was fixed with

a rigid clamp (Figure 3-3). The caudal rib was attached by

two clamps to the armature of the displacement motor (Ling

V203). A voltage was applied to the displacement motor which

moved the armature and delivered muscle stretch at varying

velocities and magnitudes.

The isolated intercostal space (ICS) was again probed for

muscle mechanoreceptor activity. When activity was observed,

the dorsal root filament was subdivided until the activity of

a single intercostal muscle mechanoreceptor was isolated. The

receptive field was localized by probing with a fine tipped

glass rod (1 mm tip diameter). The localized RMA was

characterized by the muscle spindle silent test (Figure 3-4)

(Jansen & Rudjord, 1964; Higgins & Lieberman, 1968).

Conduction velocity was obtained by electrically stimulating

the afferent receptive field and measuring the distance










0.4
WJJ




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0 0


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53
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0 0 00




4 0
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.C- *1 C 4
V 0 &n 4
0 .0 44

A ( 4 0
4) (al +

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0 g 4)cq
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45
between receptive field and recording electrodes (Matthews,

1963). Further characterization was performed during analysis

of RMA response to stretch (see below). The rib space was

then set to Linit and baseline respiratory muscle afferent

(RMA) discharge frequency recorded. The displacement protocol

was then initiated. A square wave pulse (5.0 V of varying

duration) (Grass S48) triggered a trapezoid generator

(Frederick Haer & Co. #2337) that delivered a variable

trapezoidal waveform to the displacement motor. The motor, in

turn, produced stretch of the intercostal space with varying

displacements and velocities.


Displacement Protocol


The response to intercostal muscle displacement was

determined by applying graded amplitudes of stretch to the

intercostal space. The intercostal space was initially set at

Liit, the width measured at FRC. The displacement amplitudes

were applied above Liit. The amplitudes used were: 50, 100,

200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800, and 2000 pm

(Figure 3-5). These displacements were presented at a

constant velocity of 10 gm/msec. Each displacement test was

initiated by activating the displacement motor which pulled

the caudal rib at a constant velocity to the predetermined

amplitude. The stretch was then maintained for 3 seconds,

after which the displacement motor rapidly returned the rib to








Figure 3-5. Displacement coding for an intercostal muscle spindle.
The top trace is afferent discharge. The bottom trace is the
amplitude.









Displacement Coding



. 50 r I I I I
50 pm


I11 -111 F.l_ l ll i F VI ll I. I Ii I -LJ Lli I .-.. 1.[

100 pm





1 J.Il I iJ.i II I i iii l iiiLl i II i I i I l..II J J a JI ii [ l] I .
"- -r-1-- r "T r --1--1---

200 pm





I II L I.I I II I I II.U I lrI Isec


400 pm s
---4 = I sec








Fiaure 3-5. Continued


Displacement


"-lr r


Coding



ST f rT ', I I1 44 17-1-


600 pm


14 Il i 4


itJl l L il4 lJ


44I44444


l1m"- i11"11


800--
800 urm


1 ]_ 1 1 41 1 1 111 1
I I- T r' IrlFI-tlI T II


1000 pm


|=2V


I I -- =lsec


II


44.L
.m J


1200 pm


I


*


I


*


MWX


Ml


I Ri. lF Il llIl L ll I l ,l lll, 1 .I111 l l


-~------~


- --


v-r Ii-'r -T-1- I'l-









Figure 3-5. Continued

Displacement Coding








1400 rm


4J~~44~4444~444


1600 um


I I I I I


I ill lllll ldl


1800 gm


44144444


l I li II W1I. lllll4 lll i ll.llly44


1N4P4.N4P64I


I=2V


2000 pm


I I = 1 sec


***


0


*


IN""- "- 14-Il-1" fL 'LL "i-"


I


I


'I I mil -U I 119 9 I-' I r- I ,"- ~I r i -r I


I -i | ,


" U U


1I]J r.r.l.L1 L.I 1I.1 .1111111.1 1 IL II rl.ll.L I








50

ICS Lint. There was a 10 second rest period before the next

displacement test was initiated. The discharge of the

afferent was recorded throughout the stretch.

The displacement protocol began with at least 3

displacement stretches of 2,000 im to provide a constant

stretch history in the muscle. The ICS was displaced at each

amplitude 3 times in an ascending order of amplitudes. The

RMA activity and the analogue output from the displacement

transducer were recorded on magnetic tape for subsequent

analysis.

The response of the mechanoreceptors to graded

displacement of the ICS was determined by measuring the

frequency of discharge at two different points during the

applied stretch. The first measurement was taken from a

static length of the intercostal space at Liit. This

frequency was the baseline or resting discharge of the

respiratory muscle afferent. The second discharge frequency

was determined during the stretch of the ICS, 2 seconds after

the end of the ramp phase (Figure 3-6).


Velocity Protocol


The response of RMAs to dynamic changes in intercostal

muscle length was determined by varying the stretch velocities

of the intercostal space. This experimental trial followed

the displacement test. The intercostal space was initially

set at Li.it and the velocities applied to displacements above










0) -
u u



0
QJ






4.)
o

C 4.

4) 04



N.4
04



S^' ?
0 Q4
0

4-)







lu
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5o r









0
040
4-)
4).









u 0







00



0 4) o
*4 *VH
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I 0
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44C~




















































41







41
.I.

41
I-




41


o


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a
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PQ


u


Un
4.'


p4;



41
U

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.4.


ml'"..l mrl" nrr~ln~m








53

Lint. The velocities tested were: 0.5, 1.0, 2.0, 5.0, 10,

20, 50, and 100 pm/msec (Figure 3-7). Each velocity test was

initiated by activating the displacement motor to deliver a

2000 Am displacement at the predetermined velocity. The

displacement was maintained for 3 seconds after the completion

of the velocity ramp. The caudal rib was then returned to

Linit and the muscle allowed to rest for 15 seconds. The next

velocity test was then initiated. Each velocity was tested 3

times. The discharge of the afferent was recorded throughout

the stretch. The protocol again began with a series of 3

displacement stretches to 2,000 Am to provide a constant

stretch history.

The response of the intercostal muscle afferents to

graded changes in displacement velocity was then determined.

The discharge parameters measured were the peak instantaneous,

mean ramp, static hold phase, and Linit static discharge

frequencies. The peak instantaneous frequency was obtained

during the ramp stretch of the intercostal muscle and was the

maximum frequency of discharge. The mean ramp frequency was

determined by averaging the frequencies of discharges over the

entire ramp stretch. The static Linit discharge and static

hold phase frequencies are taken from positions as described

in the displacement protocol (see Figure 3-6).








Figure 3-7. Velocity coding for an intercostal muscle spindle. (a)
Velocities of 100, 50, 20, and 10 Am/msec are shown. (b) Velocities
of 5.0, 2.0, 1.0, and 0.5 Am/msec are shown. Note the different
time scales in (a) and (b).











Velocity Coding







5.Oum/msec


2.Oum/msec


I i I it MaM l111111"i
1* 'I I jIhill


A OSum/mse se

A. .-_ =- sec


4 l


t.. .~ ..... I.I -n.l..,..l~.r...n nll~*l~s~~.L.hr.ldI I1~I(1I1 n)l)l nl(alRII


., _..








Figure 3-7.


Velocity Coding


lOOum/msec


SOum/m Il.JJ.I 11 JI J-1 se1
_ _17r|-- ... "]I|I--11F 1 r I --se[ I r


S=2V


IOum/msec


I 4--I-- = 02sec


. ,I .. I- 1. j .- t I i .lIlp _.m/1 1 1. 1 -1 I 4L1 .. 4


20um/msec


Continued


4I .. .. I II I I. .. I I..... ) { L..J I I l. l I.. I I.J .. J I ..












Analysis


Afferents were classified as Golgi tendon organs if

receptive field electrical stimulation caused the receptor to

increase its discharge frequency. Those afferents that

exhibited a pause in discharge frequency were classified as

muscle spindles. Primary muscle spindles were characterized

with the satisfaction of at least two out of three criteria:

1) a conduction velocity > 75 M/sec, 2) An increase in

discharge frequency at 100 pm displacement, 3) An abrupt

decrease in frequency discharge at the end of stretch that

increased to static hold phase levels. The data recorded on

magnetic tape were analyzed with the aid of the Spike 2

(Cambridge Electronics Design, Ltd.) computer program. The

analog signal from the displacement motor was sent from the

magnetic tape recorder to a digitizing signal processor (CED

1401). The afferent spikes were passed through a slope/height

window discriminator (Frederick Haer & Co.) and then sent to

the signal processor as timed transistorized logic (TTL)

pulses. The sampling rate of the afferent signal was 3000 Hz

to insure an acceptable error in sampling (< 3%). The program

was designed to provide the instantaneous frequency of the

afferent spikes at the corresponding amplitudes of the

displacement motor that produced the muscle stretch.








58

The frequencies used for analysis of the displacement

coding properties included the resting discharge frequency or

Linit static discharge and the static hold phase frequency.

The velocity protocol recorded these static frequencies in

addition to frequencies obtained during the dynamic phase of

stretch, the peak instantaneous frequency and the mean ramp

frequency. The program of the signal processor allowed for

the manual placement of a vertical cursor in front of a

digitized pulse. The program then computed the time interval

to the next digitized pulse (interspike interval) and

displayed the discharge frequency (frequency = 1/interspike

interval).

The data was grouped by afferent population and graphed

as means +/- Standard Error (S.E.) with the dependent variable

spike frequency and the independent variable amplitude and

velocity of muscle stretch. Scheffe's test for multiple

comparisons was used to compare the coding properties of

primary and secondary muscle spindles.













CHAPTER 4
RESULTS


Fifty-six intercostal muscle mechanoreceptors were

localized in this study. Muscle spindles accounted for 21 of

these mechanoreceptors, 10 were identified as Golgi tendon

organs, and 25 mechanoreceptors were not characterized. The

receptive field locations of these afferents were distributed

throughout the intercostal muscle (Figure 4-8). The receptive

fields of the isolated mechanoreceptors were discreetly

localized within a 3 6 mm circumferential area with a glass-

tipped probe. All receptor types were located along the

entire dorso-ventral extent of the intercostal space. The

muscle spindles, both primary and secondary were distributed

evenly along the dorsal-ventral extent of the 7th intercostal

space. The Golgi tendon organs were located primarily along

the caudal edge of the fixed anterior rib with a few isolated

along the anterior edge of the caudal rib.



Muscle Spindles


Muscle spindles were characterized as primary or

secondary afferents. Eleven receptors were identified as 10

muscle spindles and 10 were characterized as 20 muscle

spindles. Primary spindle afferents possessed higher













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62
conduction velocities than secondary endings, although there

was an overlap between the two (Figure 4-9). The mean

conduction velocity for 10 spindle afferents was 68.5 +/- 8.7

M/sec. The mean conduction velocity for 20 spindle afferents

was 43.8 +/- 6.2 M/sec. The adaptation of frequency discharge

at the end of the dynamic phase of stretch differentiated

primary and secondary muscle spindles. Those spindles that

decreased in discharge frequency gradually to static hold

phase levels were identified as being primary endings.

Secondary muscle spindles had little adaptation of the static

hold phase discharge frequency at the end of ramp stretch.


Displacement Coding 1 Muscle Spindles


Primary muscle spindles were studied with and without

intact ventral roots. The Linit static frequency was not

altered by stretch amplitudes. There was no significant

difference in the Liit static discharge frequencies after the

ventral roots were severed. Primary spindles with intact and

cut ventral roots had Lint static discharge frequencies

of 16 +/- 1 spikes/sec and 16 +/- 6 spikes/sec, respectively.

Increasing ICS displacement resulted in an increased discharge

frequency in all 10 muscle spindles studied (Figure 4-10).

The 10 muscle spindles with cut ventral roots (n=5), however,

had significantly (p < 0.05) higher discharge frequencies at

displacement amplitudes of 1200, 1400, 1800, and 2000 im.

Conversely, the mean discharge frequencies for primary








Figure 4-9. Distribution of conduction velocities for muscle
mechanoreceptors. The mean conduction velocity of primary
afferents was 68.5 +/- 8.7 M/sec. The mean conduction velocity for
secondary afferents was 43.8 +/- 6.2 M/sec. The mean conduction
velocity for Golgi tendon organs was 41.9 +/- 8.8 M/sec.







64









1.0

9 e = 10 Muscle Spindles
[ = 2 Muscle Spindles
) 8 = Golgi Tendon Organs

7 7
E-
FrI
r4
S6 ~

5 -
0


IX N- X1
Z 2
_1N


< 20 21 50 51 -80 > 80
CONDUCTION VELOCITY (M/sec)










04.)
C *i








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0 0 0 0 0 0







(oas/sa ) N kDNKflbSd --IdS
o --- 1 ---- ^'i 1 ^--- o








67

afferents with intact ventral roots (n=6) were higher (p <

0.05) than those with cut ventral roots at the lower levels of

displacement (50, 100, 200, and 400 im). Primary muscle

spindles with intact ventral roots had a discharge frequency

of 22 +/- 2 spikes/sec at 50 Am and 32 +/- 7 spikes/sec at

2000 im displacement. Primary muscle spindles with cut

ventral roots had a discharge frequency of 16 +/- 4 spikes/sec

at 50 jm and 38 +/- 5 spikes/sec at 2000 jm displacement. The

slope of the displacement frequency response for ventral roots

intact was 6.2 spikes/sec/mm and 10.4 spikes/sec/mm for

ventral roots cut. The response of one 10 spindle afferent to

displacement was recorded with ventral roots intact and then

again after the ventral roots were severed. There was no

significant difference in the Liit static discharge frequency

between the two conditions for this afferent. There was also

no significant difference in the response of this afferent to

increased ICS displacement. There was no significant

difference with ventral roots severed in either the small or

large amplitudes of displacement. Thus, the differences

observed in earlier experiments was an artifact of the

sample populations.

The response of all 10 muscle spindles to ICS

displacement were grouped. The mean Liit static discharge

frequency for all primary muscle spindles was 16 +/- 2

spikes/sec (Figure 4-11). The slope of static displacement

coding discharge frequency was found to be 8.8 spikes/sec/mm










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(oas/sayNds) ADNafnBla 3iIdS








70

(n=ll). The range of static displacement coding for primary

afferents was found to be 19 +/- 5 spikes/sec at 50 pm

displacement to 35 +/- 5 spikes/sec at 2000 pm displacement.


Velocity Coding 1 Muscle Spindles


Primary muscle spindles had a characteristic adaptation

of discharge frequency at the end of the ramp phase of

stretch. The peak discharge frequency occurred during the

dynamic phase of displacement (ramp). The peak instantaneous

frequency increased with increasing velocity of stretch. The

discharge frequencies of the afferents increased with

increasing velocities of stretch resulting in a sigmoidal

frequency-velocity relationship. The frequency discharge

pattern of a 10 spindle afferent prior to, and after ventral

root section is shown in figure 4-12. There was a decrease in

the peak instantaneous frequency (359 to 315 spikes/sec) and

mean ramp frequency (250 to 214 spikes/sec) 30 min post

ventral root section at 100 Am/msec stretch velocity that

returned to pre-ventral root section levels by 1.5 hr (315 to

350 spikes/sec). The Li,,t static discharge and static hold

phase discharge frequencies were unaffected by ventral root

section.

The range of peak instantaneous frequencies for all the

10 spindle afferents with intact ventral roots for velocities

from 0.5 100 Am/msec is 32 345 spikes/sec with a mean of

36 +/- 7 to 205 +/- 59 spikes/sec (n=6) (Figure 4-13). There








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75

was no effect of velocity on the Lint, static discharge

frequency. There was also no effect of velocity on the static

hold phase discharge frequency. The static hold phase

frequencies of 10 spindle afferents with ventral roots intact

range from 28 40 spikes/sec with a mean of 34 +/- 4

spikes/sec. The Lint static discharge frequencies of 1

spindle afferents with intact ventral roots range from 0 28

spikes/sec with a mean of 16 +/- 2 spikes/sec.

The range of instantaneous burst frequencies for 10

spindle afferents with sectioned ventral roots is 34 410

spikes/sec with a mean of 47 +/- 6 spikes/sec to 287 +/- 86

spikes/sec (n=5) (Figure 4-14). The static hold phase

frequencies for 10 spindle afferents with sectioned ventral

roots range from 32 42 spikes/sec with a mean of 36 +/- 5

spikes/sec. Primary afferents with sectioned ventral roots

had Linit static discharge frequencies ranging from 8 28

spikes/sec with a mean of 22 +/- 6 spikes/sec. For 10 spindle

afferents with sectioned ventral roots, the range of the mean

frequencies during ramp stretch was 39 345 spikes/sec with

an average of 38 +/- 6 spikes/sec to 226 +/- 64 spikes/sec.

The range of the mean frequencies during ramp stretch for 10

spindle afferents with intact ventral roots is 38 310

spikes/sec with an average of 33 +/- 9 spikes/sec to 158 +/-

38 spikes/sec. The discharge frequency response for mean ramp

stretch frequencies for 10 afferents with and without an

intact ventral roots is plotted in figure 4-15. There were no










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Figure 4-15. Primary muscle spindle afferent mean ramp discharge
frequency with and without an intact ventral roots. There was no
significant difference in discharge frequencies at the lowest
velocity of intercostal muscle stretch used (0.5 Am/msec). The
discharge frequencies for 10 muscle spindles with and without
ventral roots was 33 +/- 9 and 38 +/- 6 spikes/sec, respectively.
Primary muscle spindles with cut ventral roots were found to have
a significantly greater discharge frequency than 10 spindle
afferents with intact ventral roots. The discharge frequency for
10 spindle afferents with and without intact ventral roots was 158
+/- 38 and 226 +/- 64 spikes/sec.




















1 MS FREQUENCY DISCHARGE RANGE
350
5I|= 0.5 /m/msec
300 = 100 apm/msec


250


200 -


150


100


50


0


VENTRAL ROOT INTACT


0,

Q)







I-4
0*



rz
(73


VENTAL ROOT CUT








80

significant differences between the static hold phase or

static discharge frequencies of 10 spindle afferents with or

without ventral root section.

The range of peak instantaneous frequencies for 1

spindle afferents (n=ll) was 41 +/- 3 spikes/sec at 0.5

Am/msec velocity to 221 +/- 29 spikes/sec at 100 pm/msec

velocity. The slope was 3.0 spikes'-~"/m-'", which was the

range of greatest sensitivity. This was taken between stretch

velocities of 5 to 50 Am/msec. The static discharge hold

phase frequency for 10 muscle spindles was 35 +/- 3 spike/sec.

The Linit static discharge frequency for 10 spindle afferents

was 18 +/- 3 spikes/sec. The velocity coding profile for all

primary muscle spindles recorded to increasing intercostal

muscle stretch is shown in figure 4-16.


Displacement Coding 20 Muscle Spindles


Secondary afferents increased their discharge frequency

with increasing amplitudes of intercostal space displacement.

The Lint static discharge frequency of the 20 muscle spindles

was not affected by ventral root section (Figure 4-17).

Secondary muscle spindles with intact ventral roots (n=6) had

a mean discharge frequency of 17 +/- 3 spikes/sec at 50 Am

displacement and 22 +/- 4 spikes/sec at 2000 Am displacement.

Secondary muscle spindles with cut ventral roots (n=4) had a

mean discharge frequency of 22 +/- 7 spikes/sec at 50 Am

displacement and 32 +/- 8 spikes/sec at 2000 Am displacement.











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Secondary afferents with intact ventral roots had a mean Linit

static discharge frequency of 16 +/- 5 spikes/sec. Secondary

muscle spindles with sectioned ventral roots had a mean Linit

static discharge frequency of 19 +/- 9 spikes/sec. The

response of 20 muscle spindles (n=10) to increasing

displacement amplitudes were combined and had a slope of 3.7

spikes/sec/mm. The range for displacement frequencies for

secondary afferents was 19 +/- 2 at 50 pm to 26 +/- 3

spikes/sec at 2000 Am. The mean Linit static discharge of

these afferents was 17 +/- 2 spikes/sec (Figure 4-18).


Velocity Coding 20 Muscle Spindles


Secondary muscle spindles increased peak instantaneous

frequency with increasing velocity of intercostal muscle

stretch. The range of mean peak instantaneous frequencies for

20 spindle afferents with intact ventral roots was 20 +/- 4

spikes/sec to 81 +/- 37 spikes/sec for velocities from 0.5 -

100 Am/msec (Figure 4-19). The static hold phase frequencies

of 20 spindle afferents with ventral roots intact range from

14 28 spikes/sec with a mean of 20 +/- 5 spikes/sec. The

Linit static discharge frequencies of 20 spindle afferents with
intact ventral roots range from 0 28 spikes/sec with a mean

of 16 +/- 3 spikes/sec. The range of instantaneous burst

frequencies for 20 spindle afferents with sectioned ventral

roots (n=4) was 26 145 spikes/sec with a mean of 31 +/- 5

spikes/sec to 90 +/- 33 spikes/sec (Figure 4-20). The static










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92

hold phase frequencies for 20 spindle afferents with sectioned

ventral roots ranged from 16 38 spikes/sec with a mean of 33

+/- 5 spikes/sec. Secondary afferents with sectioned ventral

roots had Lit static discharge frequencies ranging from 12 -

27 spikes/sec with a mean of 18 +/- 6 spikes/sec. The mean

ramp stretch discharge frequency at 0.5 um/msec and 100

Am/msec for all the secondary muscle spindles (with and

without intact ventral roots, n=10) is shown in figure 4-21.

The range of the mean ramp frequencies for 20 spindle

afferents with intact ventral roots was 14 175 spikes/sec

with a mean of 20 +/- 4 spikes/sec to 74 +/- 28 spikes/sec

(n=6). The range of the mean frequencies during the dynamic

phase of ramp stretch for 20 afferents with cut ventral roots

was 20 110 spikes/sec with a mean of 25 +/- 4 to 81 +/- 30

spikes/sec (n=4).

The response of 20 muscle spindles to increased stretch

velocity were combined (Figure 4-22). Secondary muscle

spindles increased peak instantaneous frequencies with

increasing intercostal muscle stretch velocity. The range of

discharge frequency was 25 +/- 3 spikes/sec at 0.5 pm/msec to

85 +/- 21 spikes/sec at 100 gm/msec intercostal muscle stretch

velocity. Secondary muscle spindles were found to possess the

lowest sensitivity of the three types of mechanoreceptors

studied. The slope of the frequency response for 20 spindle

afferents between 5 and 100 Am/msec was 0.6 spikes-sI"/m'"".








Figure 4-21. The mean ramp frequency discharge for 2 muscle
spindle afferents with and without intact ventral roots. There was
no significant difference in discharge frequencies at the lowest
velocity of intercostal muscle stretch used (0.5 gm/msec). The
discharge frequencies for 20 muscle spindles with and without
ventral roots was 20 +/- 4 and 25 +/- 4 spikes/sec, respectively.
At the highest velocity of intercostal muscle stretch (100
jm/msec), it was found that 20 muscle spindles with cut ventral
roots were not significantly different from 20 afferents with
intact ventral roots. The discharge frequency for 20 spindle
afferents with and without intact ventral roots was 74 +/- 28 and
81 +/- 30 spikes/sec.



















2 MS FREQUENCY DISCHARGE RANGE
150
S5M= 0.5 iAm/msec
/ -= 100 /pm/msec
r-

a 100 -




S50


V 50 ,




VENTRAL ROOT INTACT VENTAL ROOT CUT









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