Citation
Respiratory Mechanoreceptor Activation of Somatosensory Cortex in Humans

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Title:
Respiratory Mechanoreceptor Activation of Somatosensory Cortex in Humans
Place of Publication:
Gainesville, Fla
Publisher:
University of Florida
Copyright Date:
2005

Subjects

Subjects / Keywords:
Breathing ( jstor )
Cerebral cortex ( jstor )
Electrodes ( jstor )
Evoked potentials ( jstor )
Mechanoreceptors ( jstor )
Neurons ( jstor )
Resistance mechanisms ( jstor )
Respiratory mechanics ( jstor )
Skin ( jstor )
Somatosensory cortex ( jstor )
City of Davenport ( local )

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University of Florida
Holding Location:
University of Florida
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All applicable rights reserved by the source institution and holding location.
Embargo Date:
5/1/2005
Resource Identifier:
74459597 ( OCLC )

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Full Text












RESPIRATORY MECHANORECEPTOR ACTIVATION OF SOMATOSENSORY
CORTEX IN HUMANS
















By

YANG-LING CHOU


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


2005




























Copyright 2005

by

Yang-Ling Chou




























This dissertation is dedicated to my beautiful homeland, Taiwan, and to my dearest
parents, Dr. I-Chang Jou and Pao-Chu Huang















ACKNOWLEDGEMENT

First and foremost, I would like to express my gratitude toward my mentor, Dr.

Paul W. Davenport, for having me as his graduate student. His enthusiastic support and

great guidance on many aspects of this dissertation were invaluable. I also gratefully

acknowledge helpful support from the rest of my committee, Dr. Daniel Martin, Dr.

Linda Hayward, Dr. Donald Bolser and Dr. Christine Sapienza, for consistently providing

me with valuable comments and suggestions during my graduate work.

My special thanks will go to Kan-Nian Hu, my very significant other whom I met

during this graduate work. His warm and thoughtful support was always my driving force

towards this graduate work. I would like to thank my father, Dr. I-Chang Jou; my mother,

Pao-Chu Huang; my two sisters, Lin-Ru Chou and Yang-San Chou, for their

encouragement and support during my graduate work.

My thanks also go to my colleagues for their thoughtful help. They are Dr.

Weirong Zhang and Camille Swartz. Their involvement was very helpful towards my

graduate work. I would also like to thank some friends for their support. They are Keng-

Liang Huang, Hsiao-Lan Sun, Dr. Chien-Hui Huang, Yuming Guo and Jeau-Jun Chen. I

always enjoy the happiness and talks we have shared together.

Finally, very special thanks go also towards all the subjects who participated in my

studies and contributed to my dissertation work. It is their cooperativeness and patience

that made such successful dissertation work.
















TABLE OF CONTENTS

page

A C K N O W L E D G E M E N T ................................................................................................. iv

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

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

ABSTRACT ........ .............. ............. ...... .......... .......... xi

CHAPTER

1 REVIEW OF CORTICAL PROJECTION OF RESPIRATORY
MECHANORECEPTORS ACTIVATION IN RESPONSE TO MECHANICAL
L O A D .............................................................................. 1

Cortical Projection of Respiratory M echanoreceptors ................................................1
R respiratory R elated Evoked Potential ............................................... .....................2
Potential Sources and Afferent Mechanisms of RREP ..........................................6
Mechanical SEP and Somatosensory Pathway ............. .............................11
Perception of M mechanical Loads ....................................... .......................14
Summary of Experiments ................... ................. ............... 18
R ationale for Study 1......................... .... .................. .. .. ........ .... ............19
R ationale for Study 2 ................................... ................. .... ... ....20
R rationale for Study 3 ................................................................... ...... 22
Sum m ary of H ypotheses.................................................. ............................... 23

2 THE CORTICAL RESPONSES TO INSPIRATORY LOADS AND TACTILE
STIMULATION FOR HEALTHY ADULTS....................................... ...............24

Introduction ............... .......... .. .................... ........................ 24
M e th o d s ..............................................................................2 8
Subj ects ............... ..... .......................... ...............28
Pulm onary Function Testing ........................................ .......... ............... 28
R R E P .............................................................................2 9
S E P .............................................................3 0
Statistical A analysis ...................................... ................ ............ 3
R e su lt ....... .. ......................................................................................... 3 1
Discussion ......... ........... ...............................32
C conclusion ................................................................................................ 36











3 CORTICAL EVOKED POTENTIALS ELICITED BY INSPIRATORY
OCCLUSION, AIR-PUFF, AND CUTANEOUS STIMULATION IN HEALTHY
A D U L T S ...................................... ..................................................... 4 5

Introduction..................................... .................................. .......... 45
M eth o d ..............................................................................................4 7
Su objects ............... ......................................................................... ......... .... 4 7
Pulm onary Function Testing ........................................ ............................... 47
RREP Elicited by Inspiratory Load ......... ................................. ....... ...............48
Mechanical Stimulation of the Chest and the Mouth....................... ...............49
Statistical A analysis .......................... ............ ..........................51
R e su lt ........... .......... .......... ......... .............................................5 1
D iscu ssion ......... ....... ......... ................................... ............................52
C onclu sion ......... ... ............... .................................... ...........................57

4 RESPIRATORY LOAD PERCEPTION AND RREP WITH INCREASED
BACKGROUND RESISTANCE ........................................ ......................... 66

In tro du ctio n ...................................... ................................................ 6 6
M eth o d ..............................................................................................6 9
Su object ................ ........................................ ............. ................ 6 9
Pulm onary Function Testing ........................................ ............................... 70
P ro c e d u re s ...................................................................... 7 0
D ata A n aly sis................................................ ................ 7 2
R e su lts ...................................... .......................................................7 3
D iscu ssio n ...................................... ................................................. 7 4
C conclusion ............................................................... ..... ..... ........ 78

5 GENERAL DISCU SSION ............................................................ ............... 84

S u m m ary o f R esu lts.............................................................................. ............... 84
D distribution of Evoked Potentials...................................... ........................... ........ 85
R R EP w ith Inspiratory O cclusion ............................................ .....................85
SEP w ith M echanical Stim ulation.................................................... .................85
Latency And Amplitude Of Short-Latency Peaks Of Evoked Potential ...................86
R R E P .......................................................................... 8 6
SE P on H an d ............................................................................... ............... 8 8
SEP on C hest and M outh.................................................. ....................... 89
Afferent Mechanisms Of Evoked Potentials ........................................................90
Sources of R R E P .................................... ............ ................ ........ .... 90
Afferent Pathways of RREP and SEP ...................................... ............... 91
Comparisons Between RREP and SEP ...................................................92
The Relationship Between RREP and Load Perceptual Process.............................93
Relationship Between RREP and Load Perception..............................93
RREP Responses to Different Load Magnitude............... .............................94
Short-Latency RREP Peak in Response to Increased Background Resistance ...94











6 C O N C L U SIO N .......... ................................................................ ..... ....... ........99

L IST O F R E FE R E N C E S ......................................................................... ................... 10 1

BIOGRAPHICAL SKETCH ........................................................................112















LIST OF TABLES


Tablege

2-1 Mean amplitude and latency data for the respiratory-related evoked potential
and the somatosensory evoked potential....................... ...... ..................38

3-1 Group mean amplitudes and latencies for P1 and N1 peaks of the RREP, mouth
SEP, and chest SEP. ................................... .. ... ....... ............ 59

4-1 Group-averaged P1, N1, and Nf latencies and amplitudes of RREP for
inspiratory resistive load and inspiratory occlusion ......................................... 79















LIST OF FIGURES


Figure pge

2-1 Respiratory-related evoked potential Schmidt ......................................................39

2-2 SEP at C4' and C3' electrode sites (C4'- Right hemisphere; C3'-Left hemisphere). 40

2-3 RREP at C4' and C3' electrode sites (C4'-Right hemisphere; C3'-Left hemisphere).41

2-4 The RREP and SEP at F4 electrode site. The Nf peak was only observed in the
RREP but not in the SEP ................. ............................ ................. ............... 42

2-5 Comparison for the amplitudes of the P1 and N1 peaks of the RREP and the
S E P .............................................................................. 4 3

2-6 Comparison between the C3' and C4' electrode sites for the P1 and N1 peaks of
the RREP and the SEP. ................................................ ................................ 44

3-1 Comparison of the RREP, Mouth SEP, and Chest SEP from frontal electrode (F4).
The black line represents the stim ulus ........................................ .....................60

3-2 Comparison of the RREP from right (C3') and left (C4') electrodes. The black
line represents the inspiratory occlusion ...................................... ............... 61

3-3 Comparison of the mouth SEP from right (C3') and left (C4') electrodes ...............62

3-4 Comparison of the chest SEP from right (C3') and left (C4') electrodes .................63

3-5 Comparison for the amplitudes of the P1 and N1 peaks of the RREP, mouth SEP
and chest SE P .........................................................................64

3-6 Comparisons between the C3' and C4' electrode sites for the P1 and N1 peaks of
the m outh SE P and chest SE P ............................................................................. 65

4-1 Nf peaks recorded from F4 electrode with different resistive loads in both no-
background resistance (a) and increased background resistance (b)......................80

4-2 P1 and N1 peaks recorded from C4' electrode with different resistive load in
both no-background resistance (a) and increased background resistance (b). ........81









4-3 P1, N1 and Nf peaks recorded from C4' and F4 electrode sites, respectively with
inspiratory occlusion (maximal resistive load) in both no-background resistance
and increased background resistance conditions................................. ...............82

4-4 Relationship between amplitude of Pl peak and changes of resistive load
magnitude with different background resistance ...........................................83

5-1 Proposed respiratory gating m odel.................................... .................................... 98















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

RESPIRATORY MECHANORECEPTOR ACTIVATION OF SOMATOSENSORY
CORTEX IN HUMANS
By

Yang-Ling Chou

May 2005

Chairman: Paul W. Davenport
Major Department: Veterinary Medicine

Humans can consciously perceive difficulty with their breathing. It is known that

humans can sense external respiratory loads by peripheral mechanoreceptors and process

the afferent information via several afferent pathways. However, neural pathways

processing the conscious sensation of respiratory loads and how this respiratory afferent

information projects to higher brain centers are still poorly understood.

The respiratory-related potential (RREP) elicited by inspiratory occlusion in

humans recorded from the scalp provided a unique way to investigate the neural

mechanisms mediating respiratory load information and the activation of

mechanoreceptor related brain activity. The major goal of this dissertation was to

investigate the activation of respiratory mechanoreceptor eliciting RREP responses to

inspiratory loads and its relation to somatosensory cortical activation. Using

somatosensory evoked potentials as a model of RREP processing, RREP responses to









inspiratory occlusion and during breathing against inspiratory resistive loads with

increased background resistance were studied in this dissertation.

The short-latency RREP components were compared with the components of the

somatosensory evoked potential (SEP) elicited by mechanical stimulation of the hand,

chest, and mouth. Short-latency components of the RREP were also compared between

no-background resistance and increased background resistance to determine the

mechanism of respiratory baseline state on afferents projecting to the cortex.

Three studies were conducted and a total of 60 healthy adults participated. Results

from the first two studies demonstrated that the RREP and SEP are both observed over

the somatosensory cortex. Unlike the SEP, the RREP appeared to be elicited by afferents

that project simultaneously to both sides of the somatosensory cortex. The RREP may

have greater amplitude due to greater population of afferents activated with a global

stimulus such as inspiratory occlusion. These results further demonstrated that the RREP

Nf peak is a unique frontal cortical activation elicited by inspiratory occlusion. The

results from the third study demonstrated the RREP can be changed by increasing the

background load and shifting the detection threshold of the subject. If the resistive load is

detectable, the RREP was present; if the load is not detectable or became undetectable

with increased background load, the RREP was absent.














CHAPTER 1
REVIEW OF CORTICAL PROJECTION OF RESPIRATORY
MECHANORECEPTORS ACTIVATION IN RESPONSE TO MECHANICAL LOAD

Cortical Projection of Respiratory Mechanoreceptors

Respiration is regulated by brainstem respiratory centers, which act as intrinsic

controllers to generate the respiratory activity. This respiratory activity can be modified

by the afferent feedback from both chemoreceptors and mechanoreceptors to meet the

respiratory demands. The automatic regulation of breathing can also be overridden by the

cognitive centers of forebrain and by conscious control of respiration. Thus, respiration is

regulated not only by brainstem automatic controller but also by cortical voluntary motor

systems (American Thoracic Society, 1999).

Regular breathing can be modified by consciously perceived sensory inputs from

changes in ventilation, such as volume, airflow, pressure, or force. It is well documented

in human studies that sensory afferents project to the somatosensory cortex (Kakigi and

Shibasaki, 1984). Thus, respiratory sensation and perception may result from the cortical

integration of respiratory afferent information. Previous studies have shown the

projections of low-threshold afferents from intercostals muscles to the cerebral cortex

(Gandevia and Macefield, 1989), and phrenic afferents to the somatosensory cortex in

both cats (Balkowice et al., 1995) and humans (Zifko et al., 1995; 1996). Cortical evoked

potentials elicited by inspiratory occlusion (Davenport et al., 1986; Revelette and

Davenport, 1990), direct vagal stimulation (Tougas et al., 1993), and pressure pulse

stimulation to the mouth (Daubenspeck et al., 2000) have been reported. These previous









works suggested that humans can consciously perceive external added mechanical loads

and lung volume changes. This conscious awareness of mechanical load implies that

there must be activation of neurons in cerebral cortex that is measurable by

electrophysiological approaches.

Balzamo et al. (1995) demonstrated that the load estimation of mechanical stimuli

applied to the middle finger was significantly lowered by the increased background

respiratory resistance in healthy humans. Activation of respiratory afferents by resistive

loads also modified the somatosensory evoked potentials elicited by median nerve

stimulation in humans (Balzamo et al., 1999). Thus, respiratory afferents activated by

mechanical loads may have similar pathways as other somatosensory modalities and may

project to the somatosensory cortex. If respiratory mechanical afferents are processed in

part by the somatosensory cortex, then neurons of the somatosensory cortex must be

activated by respiratory mechanoreceptors. In animals, afferent information processed

from both phrenic afferents and intercostals muscle afferents has been demonstrated to

activate the thalamus (Zhang and Davenport, 2003) similar to mechanosensation in other

sensory modalities (Yates et al., 1994). In addition, phrenic afferents and intercostals

muscle mechanoreceptors have been demonstrated to activate neurons of the

somatosensory cortex in cats (Davenport et al., 1985; Davenport et al., 1993). Thus,

respiratory mechanical afferents may project to the somatosensory cortex via the pathway

that is similar to other sensory modalities.

Respiratory Related Evoked Potential

Evoked potentials have been used to record the cortical neural activity elicited by a

synchronous stimulation of afferents that project to specific regions of the cortex. The

respiratory-related evoked potential (RREP) elicited by inspiratory occlusion in humans









was recorded over the somatosensory cortex (Davenport et al., 1986). The RREP was

present bilaterally in normal healthy subjects (Revelette and Davenport, 1990). The

latencies of the RREP were inversely related to the respiratory drive and the increases in

RREP amplitude were related to the magnitude of the inspiratory load (Knafelc and

Davenport, 1997). In addition, the RREP was identified bilaterally in all subjects and the

greatest amplitude of the P1 peak of the RREP was found in somatosensory cortex

(Davenport et al., 1986; Logie et al., 1998). The RREP was reported using negative

pressure mechanical stimuli, expiratory occlusions (Strobel and Daubenspeck, 1993;

Hammond and Davenport., 1999) and different timing of inspiratory stimuli (Davenport

and Revelette, 1990). Measurement of the RREP also provided a unique way to

investigate the cortical activities elicited by respiratory loaded breathing. Humans can

consciously perceive changes of airway pressure (Younes and Sanii, 1989). RREPs' were

recordings that reflected the cortical activities in response to inspiratory occlusion and

inspiratory mechanical obstruction. Thus, it is reasonable to expect that physiological

stimulation should produce a RREP with components reflective of the nature of afferent

activation of respiratory mechanoreceptors distributed throughout the respiratory system.

However, the sources of mechanoreceptors and afferent mechanisms mediating the RREP

are unknown.

The RREP elicited by inspiratory occlusion has been reported in animals

(Davenport et al., 1985; Davenport et al., 1991; Davenport et al., 2001), children

(Davenport et al., 2000), and adult subjects (Davenport et al., 1986; Revelette and

Davenport, 1990). A consistent pattern of three components of the RREP (Nf, P1, N1)

has been identified in response to occlusions presented either at the onset of inspiration or









by the mid-inspiration (Davenport et al., 1986; Davenport et al., 2000; Davenport et al.,

1996).

The greatest amplitude of the P1 peak of the RREP has been identified by using

dipole source techniques demonstrates that the generator of the P1 was bilaterally located

within primary somatosensory (Davenport et al., 1986; Logie et al., 1998). The P1

component of the evoked potential has been demonstrated to reflect the integrity of

primary sensory pathway and varies in morphology with stimulus parameters such as

intensity and frequency (Strautus et al., 1997). Results from RREP studies using different

levels of resistive loads suggested that the P1 peak is a cortical marker of the arrival of

the mechanical sensory signal to the sensory cortex (Campbell et al., 1961; Johnson,

1993; Davenport et al., 1986). These findings suggested that the somatosensory cortex is

one part of neural circuitry sub-serving respiratory load perception. In addition, Franzen

and Offenloch (1969) reported that the amplitude of SEP components was correlated with

the magnitude estimation of the stimuli. Similar results have been demonstrated in the

RREP study that the amplitude of RREP P1 peak increased as the increase of resistive

load magnitude (Knafelc and Davenport, 1997).

The N1 peak was found in the electrodes over the vertex, Cz (Davenport et al.,

1986). The N1 peak can be recorded from the vertical dipole source and is affected by

attention (Webster and Colrain, 2000). Evidence for cortical neural processing of

respiratory sensation has been identified by using the dipole modeling and concluded the

generators of N1 peak were bilaterally located within supplementary motor cortex (Logie

et al., 1998). Thus, the N1 peak may be related to the second-order afferent information









processing to the load (Davenport et al., 1996) and may be related to attention (Webster

and Colrain, 2000).

A unique negative peak, Nf, has been found at the pre-central electrodes 25-40 ms

after the occlusion (Davenport et al., 1996). This short latency frontal peak (Nf) of the

RREP represents respiratory load information processing which may be parallel to the

somatosensory P1 peak information. The Nf-P1 was hypothesized to represent parallel

activation of somatosensory cortex and prefrontal cortex (Logie et al., 1998).The short

latency Nf peak has only been found in the pre-central electrodes referenced to the joined

earlobes and the P1 peak has not been recorded from frontal electrodes (Davenport et al.,

1986), suggesting that dual cortical generators separate via thalamic pathways

(Hamalaimen et al., 1990). However, the exact neural mechanism of the Nf peak remains

unknown.

The long-latency components of the RREP reflect cognitive and psychological

states of the subject. They are influenced primarily by factors extrinsic to the physical

stimulus, such as arousal, attention, motivation, and task difficulty and are thought to be

related to the cognitive processing of respiratory load information (Bloch-Salisbury et al.,

1998; Colrain et al., 1996; Harver et al., 1995; Webster and Colrain, 2000). The RREP

P300 peak was found to exhibit changes in amplitude and latency in response to cognitive

processing of the stimulus information and perceptual factors (Bloch-Salisbury et al.,

1998; Webster and Colrain, 2000).

The role of the short-latency components of the RREP and their relationship with

other mechnosensory modalities remains unknown. Therefore, a series experiments has

been designed in the current dissertation to investigate the latencies and amplitudes of









short-latency peaks of RREP that are indicators for cortical processing of respiratory load

information.

Potential Sources and Afferent Mechanisms of RREP

Afferents in many structures of the respiratory system may contribute to the RREP

and the RREP may be the summation of activation of several afferent populations.

Respiration against mechanical loads can alter the afferent activity of mechanoreceptors

in the mouth, pharynx, larynx, lung and respiratory muscles. The inspiratory occlusion

elicits the RREP reflecting the activation of the mechanoreceptors distributed throughout

the entire respiratory system (Davenport et al., 1986).The afferent information from these

mechanoreceptors is mediated via the facial nerve, vagus nerve, superior laryngeal nerve,

recurrent laryngeal nerve, phrenic nerve, and other respiratory muscle nerves. Activation

of cortical neurons by these afferents has been reported (Davenport et al., 1986; Zifko et

al., 1996; Togus et al., 1993; Gandevia and Macefield, 1989). However, the afferents

mediating inspiratory load information and the neural process associated with loaded

breath that elicits the RREP remain uncertain.

The activation of cortical neural activity by respiratory mechanical loads has been

studied using evoked potential techniques similar to those used in recording the SEP. The

fact that subjects are consciously aware of breathing against the mechanical loads

suggests that there must be activation of neurons in the cerebral cortex.

Chou and Davenport (2004) have demonstrated that stimulation of phrenic

afferents would elicit cord dorsum potential in the dorsal horn of the cat's spinal cord. In

this study, the phrenic afferents activated by single pulse stimuli were primary group I

and II, and to a lesser extent group III afferents. Electrical stimulation of phrenic afferents

has been shown to elicit evoked potential in the area 3a and area 3b of the somatosensory









cortex (Davenport et al., 1985). In addition, cortical evoked potentials elicited by both

electrical and mechanical stimuli have been reported (Davenport et al., 1993). This study

reported that intercostal muscle afferents project to the area 3a of the somatosensory

cortex.

Stimulation of phrenic afferents activates the neurons in the ventroposterior lateral

nuclei (VPL) of the thalamus (Davenport and Reep, 1995). The stimulation of

intercostals muscle afferents activated different neurons in the VPL of the thalamus

(Davenport and Revelette, 1995). Cortical evoked potentials found in the somatosensory

cortex were associated with C5 root stimulation (Yates et al, 1994). Retrograde

fluorescent tracers in this study found in labeled cells in the oralis nuclei of the

ventroposterior complex (VPO) of the thalamus of cats suggested that phrenic afferents

project to the somatosensory cortex via thalamocortical pathway. Straus et al. (1997)

reported cortical evoked potentials elicited by phrenic stimulation in humans. In addition,

Gandevia and Macefield (1989) demonstrated cortical projections by stimulating

intercostals muscle afferents in humans. Evidence of neural substrates mediating the

afferent information from respiratory mechanoreceptors to the somatosensory cortex has

thus been provided from both animal and human studies. Therefore, information related

to respiratory mechanical activity may be transduced by respiratory mechanoreceptors

and project to the somatosensory region of the cortical cortex via a dorsal column

lemniscal pathway, which is relayed at the VPL and/or VPO of the thalamus.

Single pulse vagal nerve stimulation elicited activity in sensory cortex and

thalamus in the monkey (Hallowitz and MacLean, 1977). A similar study in cats reported

that cortical evoked potentials recorded in both the somatosensory cortex and motor









cortex elicited by vagal afferents (Kor and Massion, 1964). A positive peak of the

evoked potential with a latency of about 21-22ms was elicited by natural cutaneous

stimulation directed to the skin of the face (Schieppati and Ducati, 1984). Similarly, two

subsequent studies also showed that both facial and trigeminal nerve afferents could elicit

neural activity in the somatosensory cortex in human (Hashimoto, 1987; Seyal and

Browne, 1989). Cortical activities elicited by superior laryngeal nerve stimulation have

also been recorded in cruciate sulcus, anterior sylvian sulcus, suprasylvian sulcus and

orbital cortex in cats (Aubert and Guilhen, 1971).

There are two potential afferent pathways for lung vagal afferents: one projects to

the somatosensory and motor cortices and a second projects to the mesocortex. Although

there is limited information concerning vagal afferent pathways to the somatosensory

cortex and the neural activity elicited by vagal afferent activation in humans,

mechanoreceptor information from the upper airway and lung has been demonstrated to

elicit cortical activation in response to negative pressure (Daubenspeck et al., 2000). The

presence of both early- and late- RREP peaks in double lung transplant recipients with

and without tracheostomy suggested lung vagal afferents and upper airway

mechanoreceptors elicit a RREP in response to inspiratory occlusion but are not essential

sources of the RREP response (Davenport et al., 2004; Zhao et al., 2002; 2003). Thus,

respiratory related afferents activated by inspiratory occlusion or negative pressure pulses

elicited cortical neural activity. This information may arise from respiratory muscles, the

lung and/or the upper airways.

Breathing against an external resistive load changes the pressure, volume, and

airflow in the entire respiratory tract. These mechanical changes are mediated by









mechanoreceptors from the lung, upper airway and respiratory muscles. There are many

potential afferent sources and pathways that contribute to respiratory perception of loaded

breaths. However, it is difficult to determine the specific neural mechanism subserving

load related afferent information processing because the load dependent mechanical

events are transmitted throughout the entire respiratory tract.

Several sensory sources may contribute to respiratory sensation. Among them,

respiratory muscle afferents probably play a primary role. However, the roles of receptors

in the upper airway and lung remain equivocal. Application of external loads to the

airway increases the transluminal pressure gradient across the extrathoracic airways. This

changes the airway wall configuration and consequently modulates the sensory

information arising from those upper airways. Both animals (Davenport et al., 1985;

Davenport et al., 1993; Davenport and Hutchison, 2001) and humans (Davenport et al.,

1986; Davenport et al., 1996; Knafelc and Davenport, 1997) have been studied to

understand the afferent mechanism of the RREP. Animal studies of respiratory sensation

have been developed in dogs with tracheal stoma. These dogs were behaviorally

conditioned to detect inspiratory resistive loads and occlusions. The stoma in these dogs

excluded afferent information from the upper airway mechanoreceptors. It was found that

dogs with a tracheal stoma could reliably detect inspiratory resistive loads and occlusion

(Davenport et al., 1991). Thus, it appears that afferents in the mouth, nose, pharynx,

larynx and upper extrathoracic trachea are not mediating load detection in these dogs.

The remaining intact sources diaphragm muscle mechanoreceptors and intercostal muscle

mechanoreceptors were likely the afferents mediating the load information. Phrenic and

intercostals muscle afferents have been reported to activate neurons in somatosensory









region of the cat brain (Davenport and Reep, 1995; Davenport and Revelette, 1995).

Animal studies of the neural mechanism mediating respiratory sensations have

demonstrated that respiratory muscle afferents may be, in part, mediating respiratory

sensation (Davenport et al, 1985; Davenport et al., 1993; Davenport and Hutchison, 2001;

Zhang et al., 2003). The occlusion-elicited evoked potential, which is analogous to the

RREP in human studies, has been recorded in the somatosensory cortex of awake lambs

(Davenport et al., 2001). The RREP was present when the lambs respired via their mouth

and via an endotracheal tube.

Stimulation of intercostal muscles (Gandevia and Macefield, 1989), vagal afferents

(Tougas et al., 1993), supraglottal afferents (Daubenspeck et al., 2000), and phrenic

afferents (Straus et al., 1997; Zifko et al., 1996) activates the somatosensory cortex with

short latency evoked potentials in humans. This somatosensory cortical projection is via

the thalamus which is similar for mechanoreceptor sensation for other sensory systems.

However, the neural mechanism and afferent pathway of the RREP elicited by inspiratory

occlusion in humans remain unknown.

In summary, there may be two cortical pathways involved in respiratory central

neural processing (Davenport et al., 1995). One is the pathway for respiratory muscle

afferents. Neural information arising from respiratory mechanoreceptor enters the spinal

cord, ascends in the dorsal column, relays in the brainstem, projects to the ventroposteral

region in the thalamus via the medial lemniscal tract, and is projected through a

thalamocortical pathway to the sensorimotor cortex (Zhang et al., 2003). The function of

this pathway could relate to the mechanical events of respiratory muscles. The second

pathway involves ascending afferent information from pulmonary stretch receptors via









the vagal nerve and its branches, and possibly the upper airways mediated by the RLN,

SLN, trigeminal and facial nerves. They relay sensory information to the brainstem and

then project to the mesocortex. This circuit may be related to the behavioral aspect of

respiration.

Mechanical SEP and Somatosensory Pathway

There are receptors that inform the brain about mechanical stimuli from touch,

pressure, and vibration (Mountcastle, 1980). Cortical evoked potentials can be elicited by

different sensory modalities (Mima, 1997). Evoked potentials have been studied

extensively in humans from somatosensory modalities and provide information regarding

cutaneous potential fields generated by synchronous activation groups of neurons. In

addition, evoked potentials generated in the cerebral cortex have an inversion of polarity

as the electrode is lowered from the surface to underlying cortex; that is, a change of

polarity is recorded as an electrode passes from the positive voltage region (source

current) to the negative voltage region (sink current). An inversion of polarity of large

amplitude focal potentials thus provides strong evidence for the presence of a local

generator with dipolar properties (Wood and Allison, 1981). The activation of

somatosensory neuronal activities has been studied and well defined for its onset latency

by recording the sensory evoked potential (SEP) associated with electrical and

mechanical stimulation. Onofrj et al. (1990) concluded that short-latency SEP

components recorded following mechanical stimuli were of longer latency and smaller

amplitude than evoked potentials recorded following electrical stimuli. This result was

supported by the findings from Buchthal (1980) that more time was needed for skin

indentation and the process of receptor transduction with mechanical stimuli as compared

to electrical stimuli.









The SEP is the result of the summed activity in the somatosensory cortex in

response to somatosensory afferent input. The SEP can be expected with physiological

stimuli and can be used as a method for measuring electrophysiological and perceptual

events. Cortical surface recordings show that the early peaks of SEP were evoked only by

contralateral stimulation; ipsilateral stimulation did not produce detectable activities

(LUder et al., 1986; Wood et al, 1988; Allison et al., 1989). Studies of the SEP have

focused on the sources and components of the SEP considered to reflect both cortical and

subcortical activation of the brain (Desmedt et al., 1983, 1984; Kekoni et al., 1997). In

fact, the early components of the SEP following mechanical stimulation have been found

to correlate with stimulus magnitude and magnitude estimation and are a summation of

spatially and temporally overlapping potentials that may have separate origins

(Hashimoto et al., 1987). Since the SEP is the result of summed activity in the

somatosensory cortex in response to afferent outputs, methods can be used to quantify

both electrophysiological and perceptual events (Regan et al., 1984). The short-latency

component of the SEP elicited by mechanical stimulation was correlated with the

magnitude of the stimulus and the magnitude estimation of the subjects (FranzEn and

Offenloch, 1969; Hashimoto et al., 1992). The spatiotemporal pattern of these recordings

has been demonstrated in previous studies that a series of two early peaks recorded within

100 ms after stimulation were largest over the contralateral somatosensory cortex (Kakigi

and Shibasaki, 1984; Hashimoto, 1987). Inputs from the somatosensory cortex were

essential for activation of other cortical regions. This may explain why lesions or surgical

removal of the hand area of the human somatosensory cortex appears to abolish all

cortical SEP peaks (Stohr and Goldring, 1969; Williamson et al., 1970; Allison et al.,









1992). Patients with cerebral lesions producing abolition of contralateral short-latency

SEP also exhibit bilateral loss of long latency SEP peaks (Williamson et al., 1970).

The short-latency peaks of the SEP originate from the somatosensory cortex

located in the rostral bank of post central gyms (Allison et al., 1989a; Allison et al., 1991;

Wood et al., 1988) in humans and indicate that cutaneous mechanosensation is initially

processed in somatosensory regions of the cortex (Geyer et al., 1999). Results from direct

subdural strip recordings have shown that the occurrence of the early peaks in the

somatosensory cortex appears to play a necessary but not sufficient role in conscious

awareness of an afferent stimulus (Ray et al., 1999). Peterson et al. (1995) reported in

monkeys that short-latency components of epidural SEP elicited by median nerve

stimulation were the result of separate generators, which consisted of a number of dipole

sources in precentral gyrus (frontal electrodes).

Information related to different sensory modalities is initially transmitted along

parallel, modality-specific, multisynaptic pathways (Mountcastle, 1980). Information

detected by external sensory receptors is relayed via the pathway stations in the brainstem

to thalamus, then projected to specialized areas of the cortex. Studies of primates have

demonstrated that the primary somatosensory cortex has not only a somatotopic but also

modality-specific organization (Kaas, 1991). Area 3b and area 1 were mainly activated

by cutaneous stimulation, while area 3a receives afferent input from deep sensory

receptors and area 2 plays a somatosensory integrative function for all modalities.

Previous studies have shown the projections of intercostal muscle afferents and of the

phrenic afferents to the somatosensory cortex (Gandevia and Macefield, 1989; Davenport

et al., 1993; Zifko et al., 1995; 1996). These results support the conclusion that cortical









activation was the summation of respiratory afferents projecting to the sensory region of

the cortex.

Perception of Mechanical Loads

Mechanoreceptors activated by mechanical loads during respiration and the

pathway mediating the afferent information to higher brain center play an important role

for the perception of respiratory obstruction. Perception of respiratory mechanical events

depends on cognitive processing of afferent information from peripheral

mechanoreceptors. This is measured in conscious humans by determination of the

threshold value of a stimulus required for detection and/or the estimation of load

magnitudes over a range of stimuli above the detection threshold. Signal detection may

only require activation of a set of parallel somatosensory pathways, but magnitude

estimation involves cognitive evaluation.

Sensation of respiratory resistive loads, using psychophysical studies of load

detection and magnitude estimation of respiratory resistive load, is similar to other

mechanical load interventions to the respiratory system (Killian et al., 1980; Stubbing et

al., 1981). Psychophysical methods have been used to quantify respiratory sensation

(Wiley and Zechman, 1966). The perception of respiratory mechanical events, such as an

applied resistive load, is dependent on two processes (Johnson, 1993). The first step is

load detection, which has been studied by using difference threshold methods (Campbell

et al., 1961; Davenport et al., 1986). The second perceptual process includes

differentiation of the load type and estimation of the load magnitude. Experimentally,

subjects were required to provide an estimate of the sensory magnitude of a load above

the detection threshold using a numerical scale or cross-modality matching, such as

handgrip tension sensation to match the magnitude of respiratory sensation (Davenport et









al., 1996; Revelette and Davenport, 1990; Davenport et al., 1991). These studies showed

that the perceived magnitude of an increased extrinsic load is linearly related to the load

magnitude when a log-log transformation is used. The slope of the line is a measure of

the sensitivity of the subject to the stimulus.

Adult humans can perceive and scale respiratory mechanical loads (Bennett et al.,

1962; Buki et al., 1983; Strobel et al., 1993; Fitzpatrick et al., 1995). The mechanical

loads are added to the breathing pathway of human subjects. Resistive loads alter

respiratory pressure-flow dynamics. Added resistive loads are constructed of porous

material and are connected in series to a breathing circuit to provide linear increases in

airflow resistance. Resistive loads serve to mimic the type of internal airflow limitation

experienced by patients with obstructive lung disease such as asthma. Typically, a series

of known resistive loads are added for one or several breaths and the subject indicates if

the added load is sensed (Zechman et al., 1979). The detection of respiratory loads is

dependent on the background resistance of the subject's airways and the breathing

apparatus (Wiley and Zechman, 1966). The ability of a subject to detect an added load,

AR50, is a function of the ratio of the added load to the background load (Ro) resulting in

the Weber-Fechner Ratio, AR50/Ro. The AR50/Ro for normal subjects is 0.3, meaning that

the AR50 must be 30% above the total Ro for the load to be detected. This AR50/Ro is also

0.3 for COPD patients with elevated background resistance. The detection threshold AR50

is higher as is the Ro but the ratio remains constant.

Ventilation is a mechanical process with respiratory muscles generating the driving

force to the respiratory pump. Breathing against an external mechanical load changes the

breathing pattern in a load-specific manner. Inspiration against a suprathreshold load









results in a reduction of airflow for a resistive load (Zechman and Davenport, 1978).

Inspiratory obstruction alters the mechanical breathing process and leads to a conscious

perception of the mechanical loads. The pattern of airflow, volume and pressure changed

by adding the resistive load and the perception of the added resistive load is also changed

by adding a background load (Revelette et al., 1990). Perceiving a loaded breath is

affected by the increased background resistance. The detection threshold of increased

internal (Burdon et al., 1982) or external (Burki et al., 1978; Gottfreid et al., 1981; Wiley

and Zechman, 1966) resistance depends on the change in the background resistance

rather than the absolute value of the added load. Thus, the background resistance plays an

important role in the perception of respiratory mechanical loads. These mechanical

changes may be mediated by afferents from the mouth, airways and respiratory muscles.

The detection threshold has been determined as the load that is detected 50% of the time

(Bennet et al., 1962; Campbell et al., 1961; Wiley and Zechman, 1966). Different

threshold values have been shown to be most reliable when they are expressed as a

fraction of the background. The detection threshold has been shown to be constant in

normal subjects with externally elevated background (Stubbing et al., 1981) and

asthmatic patients with internally increased mechanical loads (Burki et al., 1978).

Breathing against mechanical loads induces changes in respiratory mechanics and there

are pathways to mediate afferent information from respiratory mechanoreceptors to the

cortex. However, the effect of the background resistance to the cortical activation of a

loaded breath is unknown.

Poor perception of respiratory effort in patients with chronic pulmonary disease

may be due to a deficit in afferent information processing originating from respiratory









mechanoreceptors or the pathways to the somatosensory cortex. It has been reported that

the perceived magnitude declines with a background resistive load of 14.2 cmH20

(Burdon et al., 1983). Decreased perception of inspiratory resistive loads occurred when a

higher background load were present to the subjects (McCloskey, 1973). It is likely that

load detection may only require the activation of the sensory pathway but the magnitude

estimation may involve evaluation of information from multiple afferent pathways.

Evidence for the somatosensory cortex involvement in the mediation of signal detection

has been investigated with the use of the EEG (Hillyard et al., 1971; Wilkinson and

Seales, 1978). They have demonstrated the relationship between evoked potentials and

measures of signal detection in different sensory modalities. Measurement of the RREP

provides a unique way to investigate cortical activity involved in the perceptual responses

to external mechanical loads.

Asthma and other chronic obstructive pulmonary diseases are often associated with

an increased airway resistance (like adding a background resistive load) and are known to

decrease the perception of added resistive loads. Perception of inspiratory loads was

blunted by the increased background airway resistance (Revelette et al., 1984). The short-

latency component of evoked potentials reflects the integrity of the primary sensory

pathways and varies in morphology with stimulus parameters such as intensity and

frequency. The long-latency components of evoked potential reflect cognitive and

psychological state of the subject. They are influenced primarily by factors extrinsic to

the physical stimulus, such as arousal, attention, motivation, and task difficulty. Although

it has been reported that the RREP can be elicited by different magnitudes of resistive

loads, no study has addressed the relationship between the effect of increased background









resistance on the conscious sensation of resistive inspiratory loads and the RREP. It is

unknown if the decreased perception of added loads in the presence of an increased

background resistance is associated with a change of RREP peaks.

Summary of Experiments

Cortical activation by respiratory stimuli has been recorded in both human and

animals. Animal studies have demonstrated that afferents in the respiratory system have

projection pathways to the cerebral cortex. Respiratory mechanical changes associated

with loaded breaths are mediated via those afferent pathways. Measurement of the RREP

provides a unique method to study cortical responses to respiratory load stimuli. The

afferent pathway, cortical distribution and neural mechanisms processing respiratory

mechanoreceptors have been only implied in human studies. However, perceptual process

related to RREP responses and the relationship between the RREP elicited by respiratory

mechanical loads and the SEP from other mechanical stimuli remains unknown. The

RREP and SEP elicited by different somatosensory modalities (negative pressure, air

puff, cutaneous stimulation) were tested in the first two parts of this dissertation. The

RREP response to resistive loads with increased background resistance was investigated

in the last part of this dissertation. If the somatosensory system is processing respiratory

perceptual information, then comparing the latencies and amplitudes of evoked potentials

elicited by respiratory load stimuli with known somatosensory stimuli will determine if

respiratory mechanosensation is processed, in part, through the somatosensory cortex.

The first two experiments were designed to determine the cortical localization and

neural mechanism of the short-latency components (with peak latency < 100ms) of the

RREP in healthy adults eliciting inspiration against mechanical loads. The short-latency

peaks of the RREP were hypothesized as indicating the initial activation of the









somatosensory cortex by breathing against the respiratory resistive load and were similar

to mechanically elicited sensory evoked potentials (Hamalaimen et al., 1990).

Rationale for Study 1

The activation of cortical neurons by respiratory mechanical loads has been studied

using evoked potential techniques similar to those used in other somatosensory systems

such as mechanical stimulation of the skin. The fact that subjects can sense the

respiratory change against loads suggests that there must be activation of neurons in the

cerebral cortex and this activation should be measurable. Therefore, the RREP provides

unique neurophysiological evidence of cortical activation during mechanically loaded

breathing. Revelette and Davenport (1990) found that the RREP was present bilaterally

and can be elicited by occlusions either at the onset of inspiration or at mid-inspiration.

They reported that the peak amplitude was greater and the peak latency shorter for the

evoked potentials produced by the mid-inspiratory occlusions. The relationship of the

RREP and mechanical load perception has been studied using graded resistive loads.

Positive correlations were found between the peak amplitude of Pl and the magnitude of

the added resistive load, and the magnitude estimation of the load (Knafelc and

Davenport, 1997; 1999). A negative peak (Nf) occurred 25-40 msec after the loaded

breath and was maximal at frontal electrode sites (Davenport et al., 1996). These

topographical data provide evidence for dipole sources both pre (motor cortex) and post

centrally somatosensoryy cortex).

It is difficult to determine the specific afferent mechanism mediating the perception

of inspiratory mechanical loads because load-dependent changes are transmitted

throughout most of the respiratory tract and pump. It has been hypothesized that the

somatosensory system is mediating respiratory perceptual information processing.









Comparing the neural markers for respiratory mechanical stimuli with known

somatosensory stimuli (skin indentation) tests if respiratory mechanosensation is

mediated, in part, by the same areas of the somatosensory cortex as mechanical

stimulation of the skin. The results of this study will provide new information on the

similarity between mechanical load neural activity related to the component peaks of the

RREP and somatosensory mechanical skin stimulation. Although one report described

the relationship between afferent activity in peripheral nerves and perception of the

evoking mechanical stimulation of receptors in hand of human subject (Knibestol and

Vallbo, 1979), it was unknown if or how the components of the SEP reflect the same

processes as the RREP. Therefore, the present study investigated short-latency

components in both the RREP and the SEP to identify the components showing a

functionally similar distribution. The hypothesis tested in this part of the research:

The RREP response to inspiratory occlusion was similar to the SEP elicited by

cutaneous mechanical stimulation on hand.

Rationale for Study 2

A cortical SEP can be recorded following air-puff stimulation of the hand

(Schieppati and Ducati., 1984; Hashimoto, 1987; Hashimoto et al., 1990), face

(Hashimoto et al., 1991) and foot (Hashimoto et al., 1992). These mechanical stimuli

physiologically activate mechanoreceptors and are specific to mechanoreceptors unlike

electrical stimulation usually employed to elicit SEPs (Gardner and Costanzo, 1980ab).

Air-puff provides reproducible tap-like stimuli without steady pressure or direct contact

to the skin. The air-puff stimulates a restricted area of skin in a spatial and temporal

pattern and is known to activate cutaneous mechanoreceptors (Hashimoto et al., 1991b).

Mouth mechanoreceptors have been proposed as one population of mechanoreceptors









mediating the RREP. If this is correct, then the air puff mouth stimulation will elicit an

evoked potential similar to the RREP. The cortical response to air-puff stimuli has been

reported in monkeys (Gardener et al, 1984) and humans (Gardner and Tast, 1981).

Therefore, air puff seems to provide an excellent method for eliciting neurophysiological

activity underlying the evoked potentials (Hashimoto et al., 1991b; Hashimoto et al.,

1992; Hashimoto et al., 1987).

The SEP elicited by mechanical stimulation exhibited a gradual increase in

amplitude and a decrease in latency with increasing stimulus intensity (Hashimoto et al.,

1992). The SEP has been recorded in response to a variety of mechanical stimuli over the

finger, palm, and face. The SEP elicited by mechanical stimuli were similar to those

elicited by inspiratory occlusion. However, RREP studies with inspiratory occlusion have

invariably documented latency delays and amplitude augmentation in early peaks relative

to cutaneous mechanical counterparts. The latency delays and amplitude augmentation

were ascribed to the global effect of mechanoreceptor activations and might be different

sensory pathways processing the information.

The second study investigated the mechanics of latency delay, amplitude

augmentation and patterns of peaks for inspiratory occlusion elicited RREP by comparing

this potential to evoked SEP with different sensory modalities from two selected areas

(chest and mouth). The chest and mouth are chosen because they have mechanoreceptors

that have a similar neural path length as respiratory muscles (chest) and they may be

activated by inspiratory occlusions to elicit the RREP (mouth). If the pattern of the RREP

is similar to that of SEP's with activation of the chest skin and mouth, then the latency









and amplitude between the RREP and SEP should be similar. The hypothesis tested in this

part of the research:

The RREP response to inspiratory occlusion is similar to the SEP elicited by

activation of mechanoreceptors on the chest wall (the level of diaphragm location, 7th

ribcage) and the buccal surface of the mouth.

Rationale for Study 3

Perception of respiratory loads is dependent of the background conditions of the

subjects' respiratory tract and is blunted by elevated background airway resistance

(Wiley and Zechman, 1966). Asthma and chronic obstructive respiratory disease is often

associated with an elevated background airway resistance and is known to decrease the

perception of added resistive loads. It is unknown if RREP peaks will decrease their

amplitude or will not be elicited in the presence of an increased background resistance.

Therefore, the third experiment investigated the short-latency RREP components elicited

by inspiratory resistive loads with and without increased background load. The aim of the

third study was to determine the RREP responses to inspiratory resistive load when

gating by the elevated background resistance, and the relationship between conscious

perceptual process of respiratory mechanical loads and RREP components. Short-latency

components of the RREP were measured in subjects under two conditions: with and

without increased background resistance. Two hypotheses were tested in this part of the

research:

Short-latency RREP components (P1, N1, and Nf) are involved in conscious

perception of respiratory mechanical loads and are recorded only when the added

resistive load is above the detection threshold, AR50/Ro is greater than 0.3.









A resistive load detectable without increased background elicits an RREP but the

RREP is not present when the background is increased to make the same load

undetectable, AR50/Ro is less than 0.3.



Summary of Hypotheses

The hypotheses are summarized below:

1. RREP response to inspiratory occlusion is similar to the SEP elicited by cutaneous
mechanical stimulation on hand.

2. RREP response to inspiratory occlusion is similar to the SEP elicited by activation
of mechanoreceptors on the chest wall (the level of diaphragm location, 7th ribcage)
and the buccal surface of the mouth.

3. Short-latency RREP components (P1, N1, and Nf) are involved in conscious
perception of respiratory mechanical loads and are recorded only when the added
resistive load is above the detection threshold, AR50/Ro is greater than 0.3.

4. A resistive load detectable without increased background elicits an RREP but the
RREP is not present when the background is increased to make the same load
undetectable, AR50/Ro is less than 0.3.














CHAPTER 2
THE CORTICAL RESPONSES TO INSPIRATORY LOADS AND TACTILE
STIMULATION FOR HEALTHY ADULTS.

Introduction

The ability of the respiratory system to maintain normal ventilation when opposed

by an external mechanical load is a motor process performed by the respiratory muscles

acting as a pump to generate the driving force for air to flow and increase the lung volume.

Application of extrinsic mechanical loads of sufficient magnitude alters this mechanical

process and leads an activation of cortical neuron activities. The development of reliable

objective neuronal methods for studying the sensory and motor mechanisms involved in

respiratory load perception has resulted in a better understanding of the mechanisms of

respiratory perception. Cortical activation by afferent stimulation is evidenced with

event-related evoked potentials (ERP). The ERP elicited by visual, auditory and

somatosensory stimuli have been well studied (Desmedt et al., 1987; Kekoni et al., 1992;

1997). The fact that subjects can sense a respiratory change against inspiratory loads

suggests there must be activation of neurons in the cerebral cortex. The activation of

cortical neurons by respiratory resistive loads has been studied using evoked potential

techniques (Davenport et al., 1986). Davenport et al. (1986) applied inspiratory occlusions

in healthy subjects and recorded the evoked potentials from the scalp. This study

demonstrated that brief occlusion of the inspiratory airway in humans produces a series of

respiratory related evoked potential (RREP) components. The RREP recorded over the

somatosensory cortex provided a unique way to investigate higher cortical activities









associated with inspiration against mechanical loads. Revelette and Davenport (1990) also

found that the RREP was present bilaterally and elicited by occlusions either at the onset of

inspiration or at mid-inspiration. They reported that the peak amplitude was greater and the

peak latency shorter for the evoked potentials produced by the midinspiratory occlusions.

Thus, the RREP provides a unique measure of respiratory load neural processing.

A consistent pattern of three peaks of the RREP (Nf, P1, N1) has been identified

(Davenport et al., 1986; Revelette and Davenport, 1990; Davenport et al., 1996; Knafelc

and Davenport, 1997). The first peak of the RREP, P1, was a positive voltage that is

elicited by the arrival of load activity from a population of afferents activated by the

inspiratory occlusion stimulus depolarizing a column of neurons in the somatosensory

cortex.

Both P1 and N1 peaks were found in the central and post-central electrodes which

correlated with the somatosensory region of the cortex (Davenport et al., 1996). The

amplitudes and location of Pl and N1 peak activity suggest that the somatosensory cortex

is one part of the neural pathway sub-serving respiratory load information processing

(Logie et al., 1998). Neither the P1 nor the N1 peak was recorded from frontal electrodes.

A negative peak, Nf, was recorded from the pre-central electrodes 25-40 ms after the

occlusion (Davenport et al., 1996). This short latency frontal peak of the RREP may be

parallel to the processing of Pl peak information.

Human skin contains receptors that inform the brain about mechanical stimuli such

as touch, pressure, and vibration (Mountcastle, 1980). The activation of somatosensory

neuronal activity has been studied by recording the sensory evoked potentials (SEP)

associated with mechanical or electrical stimuli (Onofrj et al., 1995; Desmedt et al., 1983;









1984; Deiber et a., 1986; Gardner et al., 1984; Gerber and Meinck, 2000). Onofrj et al.

(1995) reported that short-latency SEP components recorded following mechanical

stimuli were of longer latency and smaller amplitude than evoked potentials recorded

following electrical stimuli. This result was supported by the findings from Buchthal

(1980) that more time was needed for skin indentation and processes of receptor

transduction with mechanical stimuli than electrical stimuli.

Studies of the SEP have focused on the issue of sources and components of SEP

that reflect both cortical and subcortical activation of the brain (Larrson and Prevec,

1970; Desmedt et al., 1983, 1984; Kekoni et al., 1997). The SEP is the net result of

activity in the somatosensory cortex in response to the afferent inputs. The SEP is

correlated with physiological measures and used as method for measuring both

electrophysiological and the perceptual events (Franzen and Offenloch, 1969; Gescheider

and Wright, 1968; Hashimoto et al., 1988; 1992). The early components of the SEP

following mechanical stimulation were reported to correlate with stimulus magnitude and

magnitude estimation (Hashimoto et al., 1987; 1992). The activation of cortical neurons

by respiratory mechanical loads has been studied using evoked potential techniques

similar to those used in recording the SEP.

The afferents mediating load perception are primarily located in the respiratory

muscle pump (Davenport et al., 1993). However, it is difficult to determine the specific

afferent mechanism mediating the respiratory afferent information because load-

dependent changes are transmitted throughout most of the respiratory tract and pump.

The scalp was mapped for the distribution of the short latency peaks of the RREP using

the earlobe reference. The P1 peak was found predominately over the somatosensory









region of the cortex in both animals (Davenport et al., 1985; Davenport and Hutchison,

2001) and humans (Davenport et al., 1986; Logie et al., 2000). The RREP associated with

inspiratory occlusion was recorded bilaterally over the somatosensory cortex and was

also elicited by occlusions either at the onset of inspiration or at mid-inspiration

(Davenport et al., 1986; Revelette & Davenport, 1990). The P1 peak amplitude was

greater and the peak latency shorter for the evoked potentials produced by the

midinspiratory occlusions. These reports provide evidence for dipole sources both pre

(motor cortex) and post centrally somatosensoryy cortex). Thus, respiratory afferents

project via somatosensory pathways to the cerebral cortex. This is similar to

somatosensory cortical processing of cutaneous mechanical information.

Respiratory muscle afferents have transduction properties and cortical projection

pathways consistent with their hypothesized role in respiratory load sensation. Logie et

al. (1998) identified the dipole locations of the P1 peak bilaterally located within the

primary somatosensory and the Nf peak bilaterally in the supplementary motor cortex.

Activation of the contralateral sensorimotor cortex was reported following stimulation of

the intercostal muscle afferents (Davenport et al., 1993; Gandevia and Macefield, 1989)

and stimulation of phrenic afferents (Davenport et al., 1985). If this respiratory related

cortical activity is similar to evoked potentials for other sensory system, then the early P1

potential represents the arrival of the sensory signal in the somatosensory region of the

cerebral cortex.

The study of the RREP has focused on the short-latency components (P1, Nf and

N1) because they were similar to the initial sensory components in the cortical processing

of respiratory resistive load information (Davenport et al., 1986). The main function of









the somatosensory cortex is to integrate mechanical information from the skin, joint, and

muscles afferents. Both RREP and SEP elicited by mechanical stimuli represent cortical

neural activity with different scalp distributions. However, the correlation between RREP

and the mechanical induced SEP is unknown. The purpose of the present study was to

compare the inspiratory occlusion elicited RREP and the SEP elicited by cutaneous

mechanical stimulation. The SEP and RREP were recorded in the same individuals. It was

hypothesized that the P1 peak of the RREP and SEP are recorded over the somatosensory

cortex. It was further hypothesized that the latency of the SEP elicited by mechanical

stimulation is shorter than the RREP elicited by inspiratory occlusions.

Methods

Subjects

Adult subjects (9 male, 11 female; average age = 25.5) were recruited. The subjects

were healthy and normal on the basis of no self-reported history of cardiorespiratory

disease, no self-reported history of smoking and no evidence of current major or minor

illness. They were informed of the nature of the study before starting the experiment and

written consent was obtained. The institutional Review Board of the University of Florida

reviewed and approved this study.

Pulmonary Function Testing

The subjects were asked to refrain from strenuous physical activity, large meals and

caffeine for at least four hours prior to the test. Subjects were seated upright in a chair.

Spirometry testing conformed to the American Thoracic Society Standards. Forced vital

capacity (FVC), forced expiratory volume within 1 second (FEV1) and airway resistance

by the forced oscillation method were measured in each subject. A FEV1 greater than 70

% predicted was required for the subjects to continue in the study.









RREP

The subject was seated in a sound insulated room, with the back, neck and head

comfortably supported. An electrode cap with integral electrodes was used to record

scalp EEG activity. The cap was placed on the subject's head, positioned and secured

with a strap. Scalp and electrode contact was made by the application of electro-

conducing paste administered through the center opening in the electrode. The impedance

levels for each electrode was checked and maintained below 5 KQ. The electrode cap

was then connected to an electroencephalograph system (Model 12 Neurodata

Acquisition System, Grass Instruments, Quincy, MA). The EEG activity was recorded

from electrode positions; Fz, F4, F3, Cz, C4, C3, Cz', C4', C3', Pz, P4, and P3 according

to the International 10/20 system with the joined earlobes as the reference. EEG activity

was amplified and bandpass filtered (0.3Hz-1 kHz). Eye movements (EOG) were

recorded with electrodes placed over the lateral edge of the eye. The EEG activities and

mouth pressure (Pm) were recorded and led to a signal analysis system (Signal 2,

Cambridge Electronics Ltd). The experimenter and recording equipment were in the

adjacent room. The subject was monitored with a video camera during the experiment.

The subject respired through a non-rebreathing valve with the inspiratory port

connected to a pneumotachograph and occlusion value. Mouth pressure was recorded at

the center of the non-rebreathing valve, sensed with a differential pressure transducer and

amplified with a signal conditioner. The inspiratory load was presented by silently

inflating the occlusion valve at approximately the mid-inspiration point of the breath. Pm

was used for timing of respiratory occlusion. There were 2 experimental trials for the

RREP: control trial and load trial. Both trials were separated by a 5-minute rest period off

the breathing apparatus. During the load trial, inspiration was occluded for about 300









msec every 2-5 breaths. A minimum of 100 occlusions were presented. The control trial

consisted of 100 valve activations every 2-5 breaths. The subjects inspired through a side

port on the occlusion manifold and the inspiration was not obstructed with the valve

activations. The subject watched a video movie to mask the auditory cues. The RREP

was obtained by averaging a minimum of 64 occlusions. An individual occlusion

presentation was included in the average if it produced a minimum 3 cmH20 change in

Pm and there was no eye blink. The early RREP components were defined as follows: P1

was the maximal positive deflection 40-60 milliseconds following the onset of the change

in Pm. N1 was the negative deflection in the latency range of 80-120 milliseconds; Nf

was the negative deflection in the latency range of 25-40 milliseconds. Baseline-to-peak

amplitudes were determined for each peak. Latencies were determined as the time from

the onset of the Pm change to the RREP peak. The control trial used the same procedures

except there was no occluded breath.

SEP

Cutaneous mechanical stimulation consisted of a square wave indentation of the

skin on the subject's non-dominant hand. The skin indentation was produced by a

controlled linear induction motor attached to a blunt flexible tube placed 2mm above the

surface of the skin. To obtain the SEP, subjects were asked to place their forearm on a

handrest in a fixed position. The hand was placed in position with the hand and

mechanical stimulator screened from the subject by a curtain. A series of 512 cutaneous

stimulations were delivered. A strain gauge was used to calibrate mechanical pulses.

Each skin indentation had a duration of about 400 msec and was delivered every 1

second. The subject watched a videotape movie. There were 2 cutaneous stimulation

trials. The first trial was mechanical stimulation of the skin. The second trial was









activation of the stimulator but the skin was not indented. The order of the trials was

counterbalanced across subjects.

The SEP was analyzed in the same manner as the RREP. EEG activity was

recorded from scalp electrodes referenced to joined-earlobes. EOG was recorded and any

presentation containing an eye-blink was rejected. A minimum of 256 cutaneous stimuli

were averaged for each subject. Two SEP components elicited by cutaneous stimulation

were defined as follow: P1 was the maximal positive deflection 20-30 milliseconds

following the onset of skin indentation and Nlwas the negative deflection 50-70

milliseconds after the onset of skin indentation. Baseline-to-peak amplitudes were

determined for each peak of the SEP. Latencies were obtained as the time from the onset

of the indentation to the SEP peak. A total of 2 RREP and 2 SEP trials were presented in

this study.

Statistical Analysis

One-way repeated measures analysis of variance (ANOVA) was used to determine

differences between SEP and RREP components. When the ANOVA showed a

significant group difference, a multiple comparison procedures was used to test for the

differences between latencies and amplitudes for each RREP and SEP peak. The

statistical significance was determined at p < 0.05 level in this study.

Result

The RREP elicited by inspiratory occlusion was observed bilaterally and was

absent with unoccluded breaths. The P1 peak of the RREP was identified at C4' and C3'

scalp sites (Fig. 2-3). The N1 peak of the RREP was found at C4' and C3' scalp sites (Fig.

2-3). Cutaneous stimulation applied to middle finger of the subject's non-dominant hand









elicited the P1 peak at C4' scalp site (Fig. 2-2) in all subjects. The SEP N1 peak was

obtained in the C3' and C4' scalp sites (Fig. 2-2).

The P1 and N1 peaks were observed bilaterally in the RREP (Fig. 2-3) but

unilaterally in SEP (Fig 2-2). There was no significant difference in the RREP P1 peak

latency and amplitude between right (C4') and left (C3') cortices. There was no

significant difference in the RREP N1 peak latency and amplitude between right (C4')

and left (C3') cortices. The SEP P1 peak amplitude was significantly greater in the C4'

electrode site than the C3' electrode site (Fig. 2-6). The SEP N1 peak amplitude was

significantly greater in the C4' electrode site than the C3' electrode site (Fig. 2-6).

The averaged latency and amplitude for the RREP and SEP peaks are presented in

Table 2-1. The RREP peak latencies of the PI and N1 were 63.22 + 0.3 ms and 107.53 +

0.3 ms, respectively. The SEP peak latencies of the PI and N1 peaks were 27.41 + 1.3 ms

and 42.69 + 0.9 ms, respectively. The peak latencies of the PI and N1 RREP peaks were

significantly longer than the corresponding peaks of the SEP (p < 0.05). The peak

amplitudes of the P1 and N1 RREP peaks were 2.18 + 1.49 V and -3.80 + 2.1 liV,

respectively. The SEP peak amplitudes of PI and N1 were 0.56 + 0.30 iV and -0.48 +

0.23 iLV, respectively. The PI and N1 peak amplitudes of the RREP were significantly

greater than the corresponding peaks of the SEP (p < 0.05) (Fig. 2-5). The Nf peak was

observed in the RREP but not observed in the SEP (Fig 2-4). The peak amplitude of the

Nf peak was -2.51 + 1.24 V. The peak latency of the Nf peak was 25.26 + 5.1ms.

Discussion

The similarity of the RREP waveform to that observed in response to mechanically

elicited SEP was demonstrated in the present study. The P1 peak of the RREP peaks was

observed bilaterally, whereas, the analogous peak of the SEP was observed contralateral









to the stimulated hand. The scalp distributions of the RREP peaks were similar to the SEP

at the electrode positions that are over the somatosensory cortex. The latencies of the P1

and N1 peaks of the RREP were significantly longer than those of the SEP (p < 0.05).

The amplitudes of the P1 and N1 peaks of the RREP were significantly greater than those

of the SEP. The Nf peak was only observed in the frontal scalp sites of the RREP.

Inspiratory occlusion is a mechanical load that alters the afferent activity of several

populations of respiratory mechanoreceptors in the mouth, pharynx, larynx, lung and

respiratory muscles. The inspiratory occlusion halts the normal changes in inspiratory

airflow and lung volume. This change in breathing pattern results in a change in the

afferent activities throughout the respiratory system. The RREP waveform elicited by

inspiratory occlusion was similar to the SEP elicited by mechanically stimulating

cutaneous afferents. The SEP from the present study is consistent with previous studies

(Kakigi and Shibasaki, 1984; Mima et al., 1996). The latency for mechanical stimulation

SEP is slower than electrical stimulation due to the time required for skin indentation and

receptor transudation. In the present study, the RREP peaks elicited by inspiration against

an occluded airway had significantly longer latencies and larger amplitudes than the SEP

(p < 0.05). The difference between the RREP and SEP latencies may due to a longer

transduction time required for respiratory mechanoreceptors to activate the respiratory

afferents. The difference between the RREP and SEP amplitudes may be due to more

respiratory afferents activated and respiratory mechanical change activating afferents

from both sides of the respiratory tract.

The dorsal column-lemniscal system mediates the afferent pathway to the

somatosensory cortex for the SEP (Williams, 1981). The SEP is elicited by activating









afferent fibers that travel through multiple relays via the thalamus to the somatosensory

cortex (Horne and Tracey, 1979). Two ascending system may be involved to the relay of

sensory information to the somatosensory cortex: (1) the lemniscal pathway and (2) the

extra-lemniscal reticular pathway (Albe-Fessard and Bowsher, 1965). The lemniscal

pathway includes the ventroposterolateral (VPL) nuclei and ventral basal nuclei in the

thalamus. The extra-lemniscal pathway includes the brainstem reticular formation and

ventral basal nuclei in the thalamus. Jasper (1949) reported that short-latency evoked

potentials are produced by impulses traveling via the lemniscal pathway whereas; long-

latency evoked potentials are produced by impulses traveling via extra-lemniscal

pathway. The latency for the SEP in the present study is consistent with the lemniscal

pathway.

The activation of a somatosensory modality projects to the contralateral

somatosensory cortex (Kekoni et al., 1992; McLaughlin and Kelly, 1993; Nagamine et

al., 1998). Recordings of cortical responses to afferent stimuli confirm that the electrical

responses at 50 ms and 100 ms latencies are generated by activity in the SI and SII

somatosensory cortices (Hari, 1980). The SEP evoked by electrical stimulation of median

nerve at the wrist or the index finger has a similar cortical distribution (Onofrj et al.,

1990; Grisolia and Wiederholt, 1980). The SEP has been recorded with electrical

stimulation of the phrenic nerve (Zifko et al., 1995). The amplitudes of the phrenic SEP

were greatest in the postcentral gyms somatosensoryy region), which lies just posterior to

the diaphragmatic cortical representation reported by Colebatch et al. (1991). This is also

supported by the recordings from the exposed cortex in human demonstrating that the

short latency peaks of the SEP were generated in the primary somatosensory cortex









(Allison et al., 1980). The generation of short-latency SEP components reported by

Deiber et al. (1986) and Desmedt et al. (1987) demonstrated that area 3b of the

somatosensory cortex was activated by afferents from VPL via thalamocortical pathway.

Several variables have been found to alter the hand mechanical SEP. Variation in the rise

time of the stimulus alters the rate of mechanical indentation of the skin and thus the

transduction process (Johansson et al., 1979b; Jarvilehto et al., 1981). The amplitude is

dependent on the stiffness of the indentation probe, the surface area stimulated, the depth

of the indentation and the indentation duration. In the present study, a square wave rapid

onset indentation was used for focal stimulation with mild indentation depths to elicit a

reproducible cutaneous SEP.

The P1 peak of the RREP was observed to be of greatest amplitude in the C3'and

C4' electrode sites. These sites are caudal to the central sulcus (C3 and C4) and are over

the post-central gyrus, the primary somatosensory region. This is consistent with the P1

peak of the SEP elicited by mechanical stimulation of the digits (Pratt and Starr, 1981).

The P1 peak of the SEP in the present study had a latency of 20-25 milliseconds. The

recording electrode sites were over the post-central gyrus lateral to the midline which is

consistent with the somatotopical representation of the fingers in the primary

somatosensory cortex. Thus, the mechanical SEP and the RREP P1 peaks are consistent

with an activation of neurons in the primary somatosensory region of the cerebral cortex.

The N1 peak of the RREP was observed to be of greatest amplitude in the central

electrode sites. These electrode sites were over the post-central gyrus of the cerebral

cortex which is consistent with the somatotopical representation of the fingers in the

sensorimotor cortex (Pratt and Starr, 1981). In addition, Webster and Colrain (2000)









reported that the N1 peak is recorded from the vertical dipole source and is affected by

the attention. Thus, the N1 peak of RREP may be indicative of second order cortical

processing of respiratory afferent information. The N1 peak of the SEP with a latency of

40-50 milliseconds was elicited by the cutaneous stimulation. The electrode sites were

also over the post-central gyms of the cerebral cortex (Pratt and Starr, 1981). Thus, the

mechanical SEP and the RREP N1 peaks are consistent with an activation of neurons in

the sensorimotor region of the cerebral cortex.

The Nf peak was observed 25-40 ms after the onset of inspiratory occlusion and

was only found in the frontal electrode sites of the RREP. These electrode sites were over

the pre-central gyms, the prefrontal region of the cerebral cortex. Thus, the Nf peak is

unique to the RREP and may be related to the feed-forward preparation of the respiratory

behavioral motor response.

In the current study, the P1 and N1 peaks of the RREP and SEP were observed in

the same subject. The difference between the RREP PI and N1 amplitudes and the SEP

amplitudes suggests that the magnitude of neural activity is correlated with the magnitude

of stimulus. The smaller amplitude of the evoked potential recorded with cutaneous

mechanical stimulation compared to the RREP suggest that inspiratory occlusion

activates more afferents than focal mechanical stimulation of the skin.

Conclusion

Multi-channel evoked potential recordings have provided a measurement of cortical

neural activity associated activation of respiratory and cutaneous mechanoreceptors.

Mechanically stimulated cutaneous somatosensory evoked potentials has a maximal

response over the cortex contralateral to the side of stimulation. The scalp distribution of

RREP was bilateral but similar to the cortical regions of mechanically elicited SEP.









RREP P1 and Nlpeaks were found to correspond to those of the SEP. The results of the

present study suggest that the P1 peak of the RREP and the analogous P1 peak of the SEP

activated neurons in the somatosensory cortex. Thus, the P1 and N1 peaks of the RREP

are analogous to the SEP P30 and N50 peaks. The RREP, however, has a unique

activation of the pre-frontal cortex, Nf peak, which is not found in the SEP.

The exact sensory pathways mediating somatosensory processing of the respiratory

afferent information remain unknown. The differences in amplitude suggest a greater

population of respiratory afferents is activated by inspiratory occlusion than focal

indentation of the skin. The longer latencies of the RREP peaks suggest a longer time is

required for changes in airway mechanics to stimulate respiratory mechanoreceptors.









Table 2-1 Mean amplitude and latency data for the respiratory-related evoked potential
and the somatosensory evoked potential

P1 average at C3' P1 average at C4' N1 average at C3' N1 average at C4'


2.08 1.53
0.30 0.16


2.18 1.49
0.56 0.30#


-3.68 2.02
-0.33 0.17


-3.80 2.11
-0.48 0.23#


63.22 0.3
27.41 1.3


107.53 0.3
42.69 0.9


* indicates a significantly different between the RREP and SEP. # indicates a
significantly different between C3' and C4'.


Amplitude
PV
RREP
SEP


Latency
msec
RREP
SEP














Movie Digital video c mera
Movie C -
^ Pressure



SOUND ISOLATED

ROOM


RREP


Figure 2-1. Respiratory-related evoked potential Schmidt























C4'








Stimulus


0 0.1 0.2 0.3 0.4
sec



Figure 2-2. SEP at C4' and C3' electrode sites (C4'- Right hemisphere; C3'-Left
hemisphere).












C3'


Stimulus


I I I I I I I I I


0.2


Figure 2-3. RREP at C4' and C3' electrode sites (C4'-Right hemisphere; C3'-Left
hemisphere).


sec














RFAEP


5pV
F4

SEP


Stimulus


sec


Figure 2-4. The RREP and SEP at F4 electrode site. The Nf peak was only observed in
the RREP but not in the SEP.


I I I I I I I I I I







43





6-2

4-


2-








-4-

-6-


-8
RREP SEP RREP SEP
P1 P1 N1 N1



Figure 2-5. Comparison for the amplitudes of the P1 and N1 peaks of the RREP and the
SEP. The amplitude of the P1 peak of the RREP was significantly greater than
the SEP (p < 0.05). The amplitude of the N1 peak of the RREP was
significantly greater than the SEP (p < 0.05). # indicates a significantly
different between the RREP and the SEP.






44




6


4


2


'_~-0




4 -


-6
t-2

< -4


-6


-8
C4' C3' C4' C3' C4' C3' C4' C3'
RREP-P1 RREP-N1 SEP-P1 SEP-N1




Figure 2-6. Comparison between the C3' and C4' electrode sites for the P1 and N1 peaks
of the RREP and the SEP. The SEP P1 peak was significantly greater
amplitude in the C4' electrode site (p < 0.05). The SEP N1 peak amplitude
was significantly greater in the C4 electrode site (p < 0.05). There were no
significant differences in the RREP P1 and N1 peak amplitudes between the
C4' and C3' electrode sites.* indicates a significantly different between the C3'
and C4' electrode sites.














CHAPTER 3
CORTICAL EVOKED POTENTIALS ELICITED BY INSPIRATORY OCCLUSION,
AIR-PUFF, AND CUTANEOUS STIMULATION IN HEALTHY ADULTS

Introduction

Activation of mechanoreceptors by mid-inspiratory occlusion elicits the RREP

(Revelette and Davenport, 1990). Activation of the intercostal muscles (Gandevia and

Macefield, 1989), vagal afferents (Tougas et al., 1993), phrenic afferents (Straus et al.,

1997; Zifko et al., 1995), and laryngeal afferents of the upper airway (Yin et al., 1997;

Daubenspeck et al., 2000) are known to elicit cortical evoked potentials. These

respiratory afferent sources may contribute to the RREP. The early peaks of RREP are an

indicator of respiratory sensory information arriving to the cortex (Davenport et al., 1986;

Logie et al., 1998; Colrain et al., 2000). The previous study indicated a similarity in

cortical activation between the RREP and the SEP (Davenport et al., 1996; Logie et al.,

1998). There were differences in the latency, amplitude and bilateral cortical activation. It

remains unknown if these differences are due to the sources that generate the RREP, the

neural path length and/or the cortical distribution of sensory activity.

In normal adult mammalian sensory systems information from specific sensory

modalities is transmitted along parallel, modality-specific, multisynaptic pathways

(Mountcastle, 1980). The cortical projection of human mechanoreceptors has been

identified for skin mechanoreceptors. Information transduced by sensory receptors is

relayed via the spinal cord to thalamus, then project to specific areas of the cortex. The

primary somatosensory cortex has a somatotopic modality-specific organization (Kaas,









1991). Area 3b and 1 of the somatosensory cortex are mainly activated by cutaneous

stimulation, while area 3a receives afferent input from deep sensory receptors and area 2

plays a somatosensory integrative function.

The SEP elicited by mechanical stimuli was similar to those induced by inspiratory

occlusion. The SEP elicited by mechanical stimulation exhibited an increased of

amplitude and decreased of latency with increasing stimulus intensity (Hashimoto et al.,

1992). RREP studies with inspiratory occlusion have reported longer latencies and

greater amplitudes for the P1 and N1 peaks when compared with the SEP elicited by

mechanical stimuli. The latency delays and amplitude difference may be due to the

number of mechanoreceptor populations activated or may be due to different sensory

pathway processing of respiratory information.

Intercostals muscle afferents and the phrenic afferents project to the somatosensory

cortex (Gandevia and Macefield, 1989; Davenport et al., 1993; Zifko et al., 1995). In

addition, cortical evoked potentials have been elicited by vagal stimulation (Tongus et al.,

1993), inspiratory occlusion (Davenport et al., 1986; Davenport et al., 2000), and

expiratory occlusion (Hammond et al., 1999). These findings suggest that respiratory

sensation is the result, in part, of the cortical integration of multiple respiratory afferents

projecting to the somatosensory cortex.

Mechanoreceptors of the respiratory tract that can be altered by an inspiratory

occlusion are in the upper airways, the lung, the chest wall and the respiratory muscles.

The present study was designed to investigate the mechanism of the latency, amplitude

and patterns of RREP peaks. Inspiratory occlusion elicited RREP was compared to

mechanically elicited SEP with different modalities of stimulation from two selected









areas, chest wall and mouth. Thus, the purpose of this study was to compare the early

peaks of the RREP with the SEP activated by mechanical stimuli in the respiratory

related areas. If the pattern of the RREP is similar to the SEP with specific mechanical

activation, then it was reasoned that those mechanoreceptors may mediate the RREP.

However, if differences in the latencies and amplitudes occur between the RREP and the

SEP, then the mechanoreceptors in these selective areas of stimulation are not the

primary source of the RREP. Therefore, it was hypothesized that the latencies and

amplitudes of short-latency RREP components are similar to those of the mouth SEP and

chest SEP.

Method

Subjects

Twenty normal subjects were recruited (11 male, 9 female; average age = 22.35

years) for this study. Subjects were classified as normal on the basis of self-reported

habitual good health, no self-reported history of cardiorespiratory disease, history of

smoking nor evidence of current major or minor illness. Forced vital capacity (FVC) and

forced expiratory volume within 1 second (FEV1) and airway resistance were measured

in each subject. The Institutional Review Board of the University of Florida reviewed and

approved this study. The study was explained to the subject and written consent obtained.

Pulmonary Function Testing

Subjects were asked to refrain from large meals and caffeine for at least four hours

prior to the experiment. Subjects were seated upright in a chair to perform the spirometry

testing that conformed to American Thoracic Society Standards. FVC, FEV1 and airway

resistance were collected by a computerized spirometer and impulse oscillometer system.









RREP Elicited by Inspiratory Load

Subjects were seated in a sound insulated room, with the back, neck and head

comfortably supported. An electrode cap with integral electrodes was used to record

scalp EEG activity. The electrode positions were based on the International 10-20

System. The cap was placed on the subject's head, positioned and secured with a strap.

Scalp and electrode contact has been made by the application of electro-conducing paste

administered through the center opening in the electrode. The impedance levels for each

electrode were checked and maintained below 5 kQ. The electrode cap was connected to

an electroencephalograph system and EEG activity recorded. Two electrodes were placed

over the lateral edge of the eye for recording EOG. Any load presentation that occurs

with an eye-blink was discarded. The EEG activity was bandpass filtered, amplified and

led into an on-line signal averaging computer system (CED 1401, Cambridge Electronics

Ltd., Cambridge, England). The EEG activity for each presentation was stored on

computer disk. The experimenter and recording equipment were in the adjacent room.

The subject was monitored with a video camera.

The subject respired through a non-rebreathing valve with the inspiratory port

connected to the loading manifold. Mouth pressure was sensed with a differential

pressure transducer and signal conditioner. The inspiratory load was presented by silently

inflating the occlusion valve at approximately the mid-inspiration point of the breath.

Each loaded breath was separated by 2-6 unloaded breaths. A TTL pulse generated by the

inspiratory occlusion valve controller triggered the collection of 50ms of pre-trigger and

950ms of post-trigger EEG and mouth pressure (Pm) data. The duration of each occlusion

was about 200ms. There were two respiratory experimental trials for the RREP: control

trial and load trial. Both trials were separated by a 5-minute rest period off the breathing









apparatus. During the load trial, the subject was watching a pre-recorded videotape

cartoon movie with the sound masking experimental noises. One hundred loaded breaths

were presented in each trial. The control trial used the same protocol, except that there

were no occluded breaths presented.

The recorded data for each presentation were evaluated for inclusion in the

averaged RREP. The baseline EEG traces were DC corrected if necessary. The inclusion

and exclusion criteria were the same as the previous study. The presence, latency and

amplitude of RREP peaks: Nf, P1 and N1 were determined for each scalp location from

the averaged EEG traces. Peak latencies were measured as the time from the onset of the

change in mouth pressure, to the EEG peak. The zero-to-peak amplitude was measured at

the peak of each component. The RREP peaks were identified in the following manner:

P1 was defined as the positive peak within a latency window of 40-70 ms, Nf was

defined as the negative peak within a latency window of 25-40 msec, N1 was defined as

the negative peak within a latency window of 80-120 msec. The site with the largest

amplitude and shortest latency for each peak was determined.

Mechanical Stimulation of the Chest and the Mouth

Cutaneous mechanical stimulation was performed on the subject's chest wall

between the 6th and 7th ribs. This site was chosen because the nerve path length to the

cortex is approximately equal to the phrenic nerve and intercostals muscles. The skin

indentation was produced by a controlled linear induction motor attached to a blunt

flexible tube placed 2mm above the surface of the chest wall. The probe tube was in a

plastic sleeve that was placed in contact with the skin of the chest wall and fixed in

position with a harness. The mechanical stimulator was screened from the subject by a









curtain. A series of 512 cutaneous stimulations were applied by linear displacement of the

probe tube. Each indentation had a duration of 400 ms and was presented every second.

The buccal surface of the cheek was mechanically stimulated with air pulses. Air

puff stimulation has been used to elicit cutaneous SEP in humans. The air puff provides

reproducible mechanical stimuli without direct contact to the skin and permits stimulation

of a restricted area of the buccal surface. Air puff mechanical stimulation was delivered

by a tube puffing air onto the cheek of the buccal surface of the mouth. Air puff

stimulation was delivered through the tube with one end fixed in the mouthpiece, the tip

extending 2-5 mm lateral to the molars. The other end of the tube was connected to the

pressure source. The air puff had a rise time of 0.2ms and duration of 300 msec and was

driven by oxygen gas at 20 cmH20. The cross-sectional area covered by the air puff was

about 3.6 mm2. Each trial consisted of 256 stimuli delivered every second to the buccal

surface of the mouth.

During the mechanical stimulation sessions the subject watched a videotape

cartoon movie and did not attend to the stimulus. There were 2-paired sessions (4 trials)

for chest wall and mouth mechanical stimulation. The first chest trial consisted of

cutaneous mechanical stimulation. The second chest trial consisted of activation

mechanical stimulator but the skin has not been indented. The first mouth trial consisted

of positive pressure pulses applied to the buccal surface of the cheek. The second mouth

trial consisted of the activation of the positive pressure solenoid but no air was delivered.

The order of the trials was counterbalanced across subjects.

The SEP elicited by air puffs delivered to the mouth and the SEP elicited by

mechanical stimulation of the chest was analyzed in the same manner as the RREP. EEG









activity was recorded from scalp electrodes referenced to the joined earlobes. EOG was

recorded and any presentation containing an eye-blink was rejected. The SEP and RREP

peaks were identified. Both the latency and amplitude of each peak were determined for

each stimulus condition (respiration, mouth or chest).

Statistical Analysis

The descriptive statistics of all variables in the study were calculated and expressed

as mean + standard deviation (SD). One way analysis of variance (ANOVA) for repeated

measurement was conducted to compare the latencies and amplitudes of evoked

potentials. The repeated measures ANOVA was performed for each peak with different

modalities of evoked potentials. When the ANOVA showed significant differences in

peaks, a multiple comparison (Bonforoni correction) was used to test for pairwise

differences. The significant difference level of the study is set at p < 0.05.

Result

The P1, N1 and Nf peaks of the RREP were identified in all subjects (Fig 3-1. and

Fig 3-2.). The P1 and N1 peaks were identified in the mouth SEP and chest SEP (Fig 3-3.

and Fig 3-4.). The RREP elicited by inspiratory occlusion was observed bilaterally and

was absent with unoccluded breaths. The P1 peak of the RREP was identified at C4' and

C3' electrode sites (Fig. 3-2). The N1 peak of the RREP was found at C4' and C3'

electrode sites (Fig. 3-2). The P1 peak of the mouth SEP was found at C4' electrode site

(Fig 3-3). The N1 peak of the mouth SEP was found at C4' and C3' electrode sites (Fig 3-

3). The P1 peak of the chest SEP was found at C4' electrode site (Fig 3-4). The N1 peak

of the chest SEP was found at C4' and C3' electrode sites (Fig 3-4). The Nf peak was only

found in the F4 electrode site of the RREP but not I the F4 electrode site of the mouth

SEP and chest SEP (Fig 3-1).









The latencies and amplitudes for the P1 and N1 peaks of the RREP, mouth SEP and

chest SEP are presented in Table 3-1. The P1 peak latency of the RREP was 63.49 + 6.5

ms. The N1 peak latency of the RREP was 107.30 + 11.8 ms. The P1 peak latency of the

mouth SEP was 39.91 + 3.7 ms and significantly shorter than the RREP P1 peak. The N1

peak latency of the mouth SEP was 76.10 + 6.1 ms and significantly shorter than the

RREP N1 peak. The P1 peak latency of the chest SEP was 31.00 + 4.6 ms and

significantly shorter than the RREP P1 peak but not significantly different form the

mouth P1 peak. The N1 peak latency of the chest SEP was 54.17 + 2.8 ms and

significantly shorter than the RREP P1 peak but not significantly different from the

mouth P1 peak. The P1 peak amplitude of the RREP was 1.9 + 1.2 iLV. The N1 peak

amplitude of the RREP was -4.15 + 2.6 liV. The P1 peak amplitude of the mouth SEP

was 1.53 + 0.1 iV. The N peak amplitude of the mouth SEP was -2.02 + 1.4 iV. The

P1 peak amplitude of the chest SEP was 1.19 + 1.1 iLV. The N1 peak amplitude of the

chest SEP was -1.58 + 2.4 iLV. The amplitudes of the P1 peak were significantly greater

for the RREP than the chest SEP (p < 0.05). However, there was no significant difference

for the P1 peak amplitude of the RREP and mouth SEP. The N1 peak amplitude of the

RREP was significantly greater than the mouth SEP and the chest SEP (p< 0.05). There

were no significant differences for the P1 and N1 peak amplitudes between the mouth

SEP and chest SEP.

Discussion

The P1 and N1 peaks of the RREP elicited by inspiratory occlusion were analogous

to those of the SEP elicited by either air-puff stimulation to the mouth or cutaneous

stimulation to the chest. The P1 peak of the RREP was found bilaterally in the

centroparietal scalp over the somatosensory cortex. The SEP P1 peaks were also found









over the somatosensory cortex but were unilateral, contralateral to the stimulated side.

The latencies of the P1 and N1 peaks of the RREP were significantly longer than those of

the SEPs from either mouth stimulation or chest stimulation. The P1 and N1 peak

amplitudes of the RREP were significantly greater than the corresponding peaks of the

chest SEP. Only the N1 peak amplitude of the RREP was significantly greater than the

N1 peak of the mouth SEP.

Evoked potentials elicited by mechanical stimulation of the skin (Hari, 1980

Hashimoto et al., 1987) and respiratory system have been reported (Davenport et al.,

1986). However, no study has compared the cortical evoked potentials from the skin and

respiratory mechanical stimulation. The results from the present study showed that

inspiratory occlusion activates cortical neurons in the somatosensory cortex which was

also found for the SEP elicited by mouth air puff and cutaneous indentation.

The RREP P1 peak had a bilateral localization while both the mouth SEP and chest

SEP were lateralized. The P1 peak of RREP was observed to be of greatest amplitude in

the C3'and C4' electrode sites. These electrode sites are over the post-central gyms, the

primary somatosensory region. This is consistent with the reports that the P1 peak of the

RREP reflects the arrival of the afferent projection to the primary sensory area

(Davenport et al., 1986; Davenport et al., 1996). The electrode sites of the P1 peak of the

mouth SEP and chest SEP were over the post-central gyms lateral to the midline which is

consistent with the somatotopical representation of the fingers and mouth in the primary

somatosensory cortex (Pratt and Starr, 1981). Thus, the mechanical SEP and the RREP

P1 peaks are consistent with an activation of neurons in the primary somatosensory

region of the cerebral cortex. Knafelc and Davenport (1997) reported that the P1 peak









amplitude of the RREP was correlated to the magnitude of the added resistive load. This

was similar to the relationship reported by Hashimoto et al. (1988) that the peak

amplitude of the SEP elicited by air puff stimulation was correlated to the magnitude of

the stimulus intensity. Thus, the mechanically elicited SEP and the RREP may have

similar neural processing mechanism but different afferent origins.

The N1 peak of RREP was observed to be of greatest amplitude in the Cz and Cz'

electrode sites. These electrode sites were over the central sulcus at the midline of the

cerebral cortex (Pratt and Starr, 1981). The electrode sites of the N1 peak of the mouth

SEP and chest SEP were over the central sulcus at the midline of the cerebral cortex

which is also consistent with the representation in the sensorimotor cortex (Pratt and

Starr, 1981). Thus, the mechanical SEP and the RREP N1 peaks are consistent with an

activation of neurons in the sensorimotor region of the cerebral cortex. The N1 peak of

the RREP was the negative peak associated with the P1 peak of the RREP (Davenport et

al., 1986). Bloch-Salisbury et al. (1998) reported that N1 peak amplitude of the RREP

decreases as the resistive load decreased, similar to P1 peak of the RREP. The N1 peak

amplitude also increases with attention to the load. Thus, the N1 peak of the RREP may

be the second order cortical processing of the respiratory afferent information that is

based on perceiving the first-order somatosensory information and may affect subsequent

cognitive processing.

The bilateral frontal Nf peak of the RREP was not present in chest or mouth SEP's.

The Nf peak may reflect activity in prefrontal structures (Logie et al., 1998).

Thalamocortical inputs to the somatosensory cortex project predominantly to deep

cortical layers generate a surface positive initial potential (Wood and Allison, 1981;









Wood et al., 1985). The frontal Nf peak was not due to the activity in the somatosensory

cortex. It has been reported that the prefrontal cortex gated the early stage of

somatosensory input and that it exerts inhibitory modulation on sensory processing

(Yamaguchi and Knight, 1990). This may be the reason that the Nf peak was only

observed in the RREP. The Nf peak may reflect a feed-forward preparation for

compensation by a behavioral motor response, simultaneous with respiratory sensory

perception. In addition, the Nf peak generated in the prefrontal cortex may be part of the

integration of information for motor cortical output as part of the behavioral motor

response to loaded breathing (Davenport et al., 1996). The neural generators of the Nf

peak, however remain unknown.

The cortical projection of skin mechanoreceptors has been identified. Information

thansduced by sensory receptors is relayed via the spinal cord to thalamus, then project to

specific areas of the cortex. The primary somatosensory cortex has a somatotopic

modality-specific organization (Kaas and Pos, 1988).

The activation of cortical neurons by mechanical loads has been studied using

evoked potential techniques similar to those routinely used in other somatosensory

systems (Buchner et al., 1996; Rossini et al., 1996). The P1 peak followed by the N1

peak was identical in all evoked potentials with different latencies and amplitudes. Minor

differences in the extent of negative and positive fields on the scalp could be observed in

the same subject when changing the stimulus modality, but evoked potential components

and localization followed the pattern suggested by Desmedt et al. (1987).

The fundamental difference in sensory nerve conduction measured by electrical

stimulation and mechanical stimulation is that electrical stimulation activates the distal









axon and bypasses the transduction process of the receptors. Mechanical receptor

potentials are graded and sustained depolarizations of the sensory terminals and nerve

impulses are generated by this potential (Katz, 1950). Changes in receptor properties of

skin mechanoreceptors in neuropathy have been characterized by high mechanical

thresholds without significant change in receptor transduction time (Mizobichi et al.,

2002). Inspiratory occlusion, air-puff pressure pulses and chest cutaneous stimulation

used in the current study include the transduction time for the afferent mechanoreceptors,

most likely large-diameter cutaneous afferents (Johansson and Vallbo, 1979b). The

RREP and chest SEP may have similar afferent conduction distance, suggesting that time

is required when breathing against the inspiratory occlusion to bring the respiratory

mechanoreceptors to threshold. Thus, the latency differences in normal subjects between

the RREP and mechanically evoked potentials in the current study may be due to the rise

time of the mechanical stimulus inspiratoryy occlusion), force transfer process within the

respiratory system, the receptor transduction time and the conduction in the terminal axon.

The transduction time for respiratory mechanoreceptor activation during inspiratory

occlusion is unknown. In addition, the SEP elicited by air puff stimulation to the mouth

had a longer latency than the SEP elicited by cutaneous stimulation to the chest wall. This

difference may be due to the variations in the rise time of the stimulus that alters the rate

of mechanical indentation and the transduction process (Pratt and Starr, 1981).

The RREP and mouth SEP may have similar afferent sources (upper airway) and

sensory modality (pressure), suggesting that time is required when breathing against the

inspiratory occlusion to bring the respiratory mechanoreceptors to threshold. Thus, the

amplitude differences in normal subjects between the RREP and mechanically evoked









potentials in the current study may be due more mechanoreceptor populations activated

or different neural processing for the respiratory afferent information.

The SEP following mechanically air puff stimuli to the mouth had a greater

amplitude than the SEP recorded following mechanical stimulation on the chest wall. The

difference of the amplitude between the air puff stimulation and the cutaneous

stimulation may be due to a wider surface area stimulated with air pressure, greater

receptor density and/or differences in sensory amplification by second order neural

processing system. In addition, Hashimoto et al. (1992) reported that the SEP elicited by

air puff stimulation exhibited an increased amplitude and decreased latency with

increasing intensity. Thus, the amplitude differences between the air puff and cutaneous

stimulation may be due to different stimulus intensity. However, it is difficult to compare

the intensity between two different sensory modalities (pressure versus skin indentation).

Conclusion

In summary, the observation of the evoked potentials elicited by different types of

mechanical stimulation demonstrated that: 1)The inspiratory occlusion may activate

mechanoreceptors in the mouth, upper airway, lower airway, lung, and respiratory pump

muscle; 2) These mechanoreceptors may activate cortical neurons in the somatosensory

region of the cerebral cortex; 3) The P1 and N1 peaks of the RREP elicited by

interrupting occlusion are analogous to the P1 and N1 peaks of the SEP elicited by air

puff or cutaneous mechanical stimulation.; 4) a similar temporal activation sequence for

the P1 and N1 peaks was observed for the RREP and the SEPs; 5) The Nf peak was only

elicited by inspiratory occlusion; 6) The RREP P1 and N1 peak latencies were longer

than the corresponding peaks of the mouth SEP and chest SEP; 7) The P1 and N1 peak

amplitudes of the RREP were significantly greater than those of Chest SEP; 8) The N1









peak amplitude of the RREP was significantly greater than the mouth SEP; 9) There were

no significant difference between the chest SEP and mouth SEP for the P1 and N1 peaks;

10) The P1 dipole sources were located in the contralateral somatosensory area for the

mouth and chest SEPs and bilaterally in the same somatosensory cortical region for the

RREP. Thus, it is concluded that the RREP elicited by inspiratory occlusion processes

respiratory mechanical information from multiple, bilateral afferent sources.

Mechanically elicited SEPs project to similar regions of the somatosensory cortex.

Cortical activation elicited by respiratory mechanical loads is evidenced by RREP

recordings. The cortical neurons activated by respiratory mechanical loads elicit the

RREP similar to the SEP for other somatosensory systems. This suggests that respiratory

mechanoreceptor-activated cortical neurons are in the somatosensory cortex and the

information is mediated through somatosensory pathways. The location of the

mechanoreceptors and neural mechanism involving the respiratory sensation remain

uncertain.









Table 3-1


Group mean amplitudes and latencies for P1 and N1 peaks of the RREP, mouth
SEP and chest SEP


P1 C3' P1-C4' N1-C3' N1-C4'

Amplitude ([tV)
RREP 1.89 + 1.40* 1.90 + 1.23* -3.90 2.01* -4.15 2.55*
Mouth-SEP 0.94 + 0.76 1.53 + 0.91t -1.58 + 1.45 -2.02 + 1.39
Chest-SEP 0.56 0.62 1.19 1.1t -1.25 2.05 -1.58 2.41
Latency (msec)
RREP 63.49 6.53*s 107.30 11.82*s
Mouth-SEP 39.91 3.71 76.10 6.08
Chest-SEP 31.00 4.55 54.17 2.76


* indicates a significant difference between RREP and chest SEP.
Indicates a significant difference RREP and mouth SEP.
f indicates a significant difference between right and left cortices.








60








-4 RREP
-2


N 2 Nf



Mouth SEP







Chest SEP
-4 -
-4














Figure 3-1. Comparison of the RREP, Mouth SEP, and Chest SEP from frontal electrode
(F4). The black line represents the stimulus. The Nf peak was only observed
in the RREP elicited by inspiratory occlusion.















-1-
0-
1-
2-
3-

-2-
-1 -


N1

P1



I ,


I
200


Mvv


I
400


I
600


msec
Figure 3-2. Comparison of the RREP from right (C3') and left (C4') electrodes. The
black line represents the inspiratory occlusion. P1, which is the positive peak
of RREP, represented by black arrow; N1, which is the negative peak of
RREP, represented by gray arrow.







62





-2-
-1

<2


3-

P1
-2-




2 -A 0


I I I I
0 100 200 300
msec


Figure 3-3. Comparison of the mouth SEP from right (C3') and left (C4') electrodes. The
black line represents air-puff stimulus. P1 is the positive peak of the mouth
SEP (black arrow). Nlis the negative peak of the mouth SEP (gray arrow).






63




-3-






0

1- N1

2-1P
P1

-2-









I I I I I I
-100 0 100 200 300 400
msec



Figure 3-4. Comparison of the chest SEP from right (C3') and left (C4') electrodes. The
black line represents the cutaneous stimulus. P1 is the positive peak of the
chest SEP (black arrow). NI is the negative peak of the chest SEP (gray
arrow).











6


4 *


2








-4


-6
*#
-8

P1 P1 P1 N1 N1 N1
RREP Mouth Chest RREP Mouth Chest
SEP SEP SEP SEP

Figure 3-5. Comparison for the amplitudes of the P1 and N1 peaks of the RREP, mouth
SEP and chest SEP. The amplitude of the P1 peak of the RREP was
significantly greater than the mouth SEP (p < 0.05). The amplitude of the P1
peak of the RREP was significantly greater than the chest SEP (p < 0.05).The
amplitude of the N1 peak of the RREP was significantly greater than the chest
SEP (p < 0.05). There were no significant difference between the P1 peak of
the mouth SEP and chest SEP. There were no significant difference between
the N1 peak of the mouth SEP and chest SEP. indicates a significant
difference between the RREP and chest SEP amplitude.# indicates a
significant difference between the RREP and mouth SEP.












4

*
*

2



-L0




-2 I




-4 -




C4'-P1 C3'-P1 C4'-N1 C3'-N1 C4'-P1 C3'-P1 C4'-N1 C3'-N1
Mouth Mouth Mouth Mouth Chest Chest Chest Chest
SEP SEP SEP SEP SEP SEP SEP SEP


Figure 3-6. Comparisons between the C3' and C4' electrode sites for the P1 and N1 peaks
of the mouth SEP and chest SEP. The mouth SEP P1 peak was significantly
greater amplitude in the C4' electrode site (p < 0.05). The chest SEP P1 peak
was significantly greater amplitude in the C4' electrode site (p < 0.05). *
indicates a significantly different between the C3' and C4' electrode sites.














CHAPTER 4
RESPIRATORY LOAD PERCEPTION AND RREP WITH INCREASED
BACKGROUND RESISTANCE.

Introduction

The inspiratory airflow is impeded by applying resistive loads during breathing.

The breathing pattern during a resistive loaded breath decreases (more negative) mouth

pressure as airflow increases, both parameters peak at mid-inspiration. When the

magnitude of the resistive load increased, airflow decreases further due to the impedance

imposed by the resistive load (Noble et al., 1972). As the resistive load magnitude

increases, more negative pressure is needed to overcome the resistance. When the

resistive load magnitude reaches a sufficient level, cognitive awareness (detection) of the

load occurs. Load detection (LD is studied using techniques to measure the ability of

subjects to signal detection of added loads to breathing. The ability of a subject to detect

an added load, AR, is a function of the ratio of added load to the initial background load

(Ro), called the Weber-Fechner fraction, AR/ Ro. The detection of resistive loads is

dependent of the background resistance of the subjects' airways and the breathing

apparatus. The detection threshold is defined as the AR that is signaled as detected for 50

% of the load presentation, AR50. The threshold for detection of resistive loads has been

reported to be a constant fraction AR50/ Ro of about 0.3 for normal subjects and patients

with respiratory disease. The perception of respiratory mechanical events depends on

cognitive processing of afferent information from mechanoreceptors in the respiratory

system. Bennett et al. (1962) reported a variable detection threshold AR50 for resistive









loads in healthy adults of 2-4 cmH20/f/sec. Wiley and Zechman (1966) reported that the

relationship between the background resistance and the detection threshold for a various

background load conditions was 0.25-0.3 despite a large variability in the AR detection

threshold values. The variability in the detection threshold value among these studies was

thus due to the variation in the background resistance, which includes both the subject's

intrinsic respiratory resistance and the resistance of the apparatus.

Killian et al. (1980) compared the detection threshold when the resistive loads were

applied at different inspiratory flows with different background resistances. They

suggested that the resistive load was modulated by the relationship between pressure and

airflow over the early part of inspiration, rather than changes of pressure alone. Zechman

et al. (1985) reported that transdiaphragmatic pressure (Pdi) increased significantly just

before detection of added loads without change in airflow or volume. They concluded

that load detection occurred when the change in Pdi reached a critical level. This study

demonstrated that there was a transdiaphragmatic threshold for load detection and

implicated that the respiratory pump may be a source for the afferent signals of load

detection (Zechman et al., 1985).

The pattern of airflow, volume and pressure changed by adding resistive loads

and the perception of the added resistive load was also changed by adding the

background load (Revelette et al., 1990). These mechanical changes may be mediated by

afferents from the mouth, airways and respiratory muscles. Multiple respiratory sensory

mechanisms may contribute to the respiratory sensation when breathing against a

mechanical load. Breathing against mechanical loads may induce changes in respiratory









mechanics and there must be pathways to mediate afferent information projection from

respiratory mechanoreceptors to reach the cortex.

Poor perception of respiratory loads in patients with chronic pulmonary disease

may be due to altered processing of afferent information from respiratory

mechanoreceptors. It has been reported that the perceived load magnitude declines with a

background resistive load of 14.2 cmH20 (Burdon et al., 1983). Decreased magnitude

perception of inspiratory resistive loads occurred when a higher background load was

present (McCloskey, 1973). Thus, it is likely that load detection may only require the

activation of the sensory pathway while magnitude estimation may involve evaluation of

information from multiple afferent inputs.

The short-latency components of evoked potentials reflect the integrity of the

primary sensory pathways and vary with stimulus parameters such as intensity and

frequency (Nagamine et al., 1998). The long-latency components of evoked potentials

reflect cognitive and psychological state of the subjects (Webster and Colrain, 2000).

Inspiratory occlusion produces a series of short-latency RREP peaks, P1 and N1 peaks

recorded from electrodes overlying the somatosensory cortex and Nf identified over the

frontal region of the cortex (Davenport et al., 1996; Webster and Colrain, 2000).

Inspiratory occlusion of the respiratory system elicited the RREP reflecting the activation

of respiratory mechanoreceptors (Davenport et al., 1986). Dipole sources for the P1 and

Nf peaks were bilaterally located within primary somatosensory and supplementary

motor cortices, respectively (Logie et al., 1998). However, the pathway of respiratory

loaded information to the cortex is unknown.









Knafelc and Davenport (1997) demonstrated a relationship between the amplitude

of the P1 peak of the RREP and the perceived magnitude of inspiratory resistive loads.

They reported that the P1 peak amplitude of the RREP was a function of the perceived

load and proportional to the magnitude of the resistive load. However, the relationship

between the effects of increased background resistance on the RREP is known. If the

RREP is associated with perception of added loads then the addition of an increased

background resistance should affect the RREP. Therefore, the purpose of the present

study is to determine the effects of background load on RREP responses to inspiratory

resistive loads. It was hypothesized that short-latency components (P1, N1, and Nf) of the

RREP are involved in conscious perception of respiratory mechanical loads and are

recorded only when the added resistive load is above the detection threshold of the

subject. It was further hypothesized that a resistive load detectable without increased

background resistance may have an RREP but the RREP may not be present when the

background is increased to make the same load undetectable.

Method

Subject

Subjects (10 men, 10 women, averaged age= 25.8 yr) with no history of

cardiovascular disease, neurological disease, no history of smoking and no evidence of

current major illness participated in the study. The Institutional Review Board of

University of Florida reviewed and approved the protocols used in this study. All subjects

were informed of the nature of the study before starting the experiment and written

consent was obtained.









Pulmonary Function Testing

All subjects were seated upright in a chair to perform a pulmonary functional test

that conformed to American Thoracic Society Standards. The subjects must have an

FEV1 greater than 70% predicted to continue in the study.

Procedures

The subject was seated comfortably in a lounge chair in a sound-isolated chamber,

separated from the experimenter and experimental apparatus. A standard set of

instructions was presented to the subjects to inform them of their task. The subject

respired through a mouthpiece and nonrebreathing valve (Hans Rudolph, 2600 series,

Kansas City, MO) connected to a loading manifold. The loading manifold was hidden

from the subject's view and connected to a pneumotachograph (Hans Rudolph, Model

4813) attached by reinforced tubing to the inspiratory port of the nonrebreathing valve.

The resistive loads were sintered bronze disks placed in series in the loading manifold

and separated by stoppered ports. Mouth pressure (Pm) was recorded from a port in the

center of the nonrebreathing valve. The Pm was displayed on an oscilloscope for timing

the load applications and stored on a computer (Model 1401, Cambridge Electronic

Design).

An electrode cap with integral electrodes was used to record scalp EEG activity.

The electrode positions were based on the International 10-20 System. The cap was

placed on the subject's head, positioned and secured with a strap. Scalp and electrode

contact was made by the application of electro-conducing paste administered through the

center opening in the electrode. The impedance levels for each electrode was checked and

maintained below 5 KQ. The electrode cap was then connected to an EEG system (Model

12, Grass instruments, Quincy, MA). EEG activity was monitored with an oscilloscope









monitor. The EEG activities were recorded and referenced to the joined earlobes. Two

electrodes were placed over the lateral edge of the eye for recording vertical

electrooculograms (EOG). Any load presentation that occurred with an eye-blink was

discarded. The EEG signals were band-pass filtered (0.3 1 KHz), amplified and

sampled (2 kHz) for computer analysis. The EEG activities were led into signal averaging

computer system (Model 1401, Cambridge Electronics Design).

All subjects were instructed to relax all postural and facial muscles and to breathe

as normally as possible. The subject wore a nose clip and breathed through a mouthpiece

connected to the non-rebreathing valve and loading apparatus. The subject inspired

through the minimum resistance port of the loading manifold. The occlusion valve was

connected to the opening of this minimum resistance port. The inspiratory load was

presented by silently inflating the occlusion valve at approximately the mid-inspiratory

point of the breath. Activation of the occlusion valve closed this port and channeled the

inspired air through one of three manifold resistance ports. When a resistance was

presented, the stopper for that port was removed. Activation of the occlusion valve on the

minimal resistance port was then closed and the subject inspired through the increased

resistance. The inspiratory resistive load was presented as an interruption of inspiration.

The balloon pressure was used to trigger the computer for data sample collection. Each

load was separated by 2-6 unloaded breaths. The load was applied for a duration of 500 ms.

Previous results from this laboratory have shown that the averaged detection threshold value

for resistive loads in healthy subjects with no background load is 1-2 cmH20/f/s. One

resistive load that was below the detection threshold and two resistive loads that exceed the









detection threshold were used for eliciting the evoked potentials (R1=0.2, R2=3.8, and R3=

23.3 cmH20/f/s).

Two trials were presented: 1) without elevated background resistance and 2) with an

elevated background resistance of 13.3 cmH20/f/s. Each trial was separated into two sets

of load presentations. Each set was separated by a rest period off the breathing apparatus.

The order of the presentation of the three resistive loads was randomized to minimize

temporal, order and sequence effects. Each set consisted of 120 presentations. Within

each set of 120 load presentations were tested by using 24 blocks of five presentations of

each load. Ordering of the different presentation blocks was randomized, with the

restriction that each successive set of 3 blocks contained only one block of each resistive

load level. Thus, over the set of 120 presentations each block of five trials of a given load

was presented 8 times (40 total presentation of each load magnitude). The randomization

was independently drawn for each set of 120 trials and for each subject with the 2 sets of

presentations. Thus, each load magnitude was presented 80 times. The time required to

present the entire study was -3 hours, including subject preparation time. For 5 of the 20

subjects, an additional experiment was performed with inspiratory occlusion replacing R3.

Data Analysis

The Pm and EEG activity for each load presentation were digitized and stored on

the computer for subsequent signal averaging (Signal 2, Cambridge Electronics Design).

Each resistive load magnitude was averaged separately. The data analysis was performed

for each load magnitude including the loads from the two trials.

Individual subject files were used for RREP peak component identification,

amplitude and latency analysis. Baseline-to-peak amplitude values were determined

separately for each peak. Peak amplitudes were correlated with the resistive load









magnitudes. The P1 peak is the positive potential occurring within a 40-70 ms time

window. The N1 peak is the negative potential occurring between 80-120 ms. The Nf

peak is the negative peak in the frontal electrodes occurring at 25-40 ms. The latency and

amplitude of each RREP peaks (P1, N1 and Nf) were compared using one-way, repeated-

measures analysis of variance (ANOVA). A p < 0.05 was considered as statistically

significant.

Results

P1, N1, and Nf peaks were present in all subjects with no-background resistance for

R2 and R3 loads (Fig 4-la and Fig 4-1b). With increased background resistance, PI, N1

and Nf peaks were present in all subjects with the increased background load only for the

R3 load and inspiratory occlusion (Fig 4-2a and Fig 4-2b). There was no significant

effect of background resistance for the latencies of the P1, N1, and Nf peaks of the RREP

for each inspiratory resistive load, and occlusion when present (Table 4-1).

Mean amplitudes of the P1, N1 and Nf peaks for each inspiratory resistive load

and occlusion are presented in Table 4-1. P1 and Nf peaks showed a significant main

effect of resistive load magnitudes with no-background resistance. The P1 peak

amplitude showed a significant difference between no-background resistance condition

and increased background resistance condition (Fig 4-3).

The AR50 remained the same but Ro changed to Ro' when increasing the

background resistance. Thus, the baseline background resistance was Ro= 4.8

cmH20/f/s, averaged resistance of the subject and apparatus with no-background load.

The background resistance was increased to Ro'= 16.4 cmH20/f/s, the resistance

consisted of the subject and 13.3 cmH20/f/s increased background resistance. The ratio

(AR5o/Ro) is 0.3 for normal subjects. R2/Ro is changed with the increased background









resistance increased to 0.1 and becomes undetectable. There was no P1 peak present with

R2. R3/Ro was not changed with the background resistance increased and was detectable

as it was above 0.3. Thus, P1 peak amplitude of the RREP is the function of the detection

threshold (Fig 4-4).

Discussion

The RREP peaks, P1, N1, and Nf were present with detectable resistive load

magnitudes (R2 and R3) and were not present with the load (R1) below the detection

threshold for the no-background resistance trials. In contrast, P1, N1, and Nf peaks were

absent with R1 and R2 loads below the detection threshold with increased background.

All the peaks of the RREP were present with R3 load, which was above the detection

threshold with increased background resistance. These results demonstrated that the P1,

N1 and Nf peaks are related to load perception and changes in perception changes the

RREP peaks.

Inspiratory resistive loads require the inspiratory muscles to overcome the load to

generate airflow (Wiley and Zechman, 1966). The increased tension developed by the

inspiratory muscles may be the stimulus mediating the perceived magnitude of added

loads (Killian et al., 1980; Killian et al., 1984). Resistive loads are airflow-dependent

loads and a decrease in the inspiratory airflow reduces the pressure changes associated

with the increased load. The decrease in these load-related mechanical parameters with a

decreased inspiratory effort can result in a reduced sensory stimulation and

underestimation of the load magnitude. The ventilatory response to external mechanical

loads depends on the type of load (Younes et al., 1994). It has been reported that both

minute ventilation and mean inspiratory flow rate decreased when breathing against a

resistive load in conscious man (Dressendorfer et al., 1977). The changes in the breathing









pattern compensate for the increased work of breathing (Zechman et al., 1985). However,

the afferent mechanisms involved in the behavioral load compensation responses remain

unclear.

When a stimulus of constant intensity is applied, the sensation it produces has two

stages. The first stage consists of an increased sensory magnitude; during the second

stage the sensation diminishes in magnitude over time as accommodation produces

adaptation to the load. The afferent pathway mediating the load detection has not been

identified; however, the receptors within the lung, chest wall, and upper airways may be

involved in respiratory sensation (Davenport et al., 1991; Knafelc and Davenport, 1997;

Harver, 1987). The detection of an inspiratory load is a complex chain of events

involving cortical sensory processing of respiratory mechanoreceptor activity (Kifle et al.,

1997; Fritz et al., 1999). Burdon et al. (1983) reported that perceptual performance was

not altered by increased background resistance. It is likely they failed to demonstrate an

effect of increased background load in scaling performance because their protocols

resulted in decreased airflow and loss of stimuli with increased background resistance.

Revelette et al. (1984) reported that the shift in the exponent for magnitude estimation

with increased background resistance is associated with an increase in the load detection

threshold. This is consistent with our findings in the present study that the resistive load,

below the detection threshold with the increased background load will not elicit RREP.

The activation of cortical neural activity by resistive loads has been studied using

evoked potential techniques (Knafelc and Davenport, 1997). The RREP elicited by

inspiratory occlusion applied at mid-inspiration was recorded from the somatosensory

region of the cortex in healthy adults (Davenport and Revelette, 1990; Davenport et al.,









1986). Early peaks of the RREP were identified in healthy adults, asthmatics and double

lung transplant recipients (Davenport et al., 1996; Kifle et al., 2000; Zhao et al., 2003).

The P1 peak reflects the depolarization of cortical neurons by the arrival of afferent

activity stimulated by the occluded breath.

Franzen and Offenloch (1969) reported that the magnitude of the early positive

peak of the touch elicited somatosensory evoked potential (SEP) was correlated with

mechanical stimulus amplitude and magnitude estimation of the touch. If the RREP is

analogous to the SEP, then the amplitude of the P1 peak of the RREP should correlate

with the load magnitude. This is consistent with the result from Knafelc and Davenport

(1997) reporting a relationship between the RREP P1 peak amplitude and resistive load

magnitude. They reported that there was a log-log relationship between the magnitude of

the resistive load and P1 peak amplitude when a series of resistive loads were applied

(Knafelc and Davenport, 1997, 1999). However, it remained unknown if the threshold for

eliciting the RREP corresponded to the detection threshold. The relationship between the

RREP P1 peak amplitude and the resistive load magnitude was found in the present study

that the relative afferent signal strength would be changed by increasing the background

resistance. Thus, the RREP peaks were present or absent depending on the detectability

of the load. If the load was above the detection threshold, the RREP was present.

However, if the load was below the detection threshold, the RREP was absent. Moreover,

changing the detection threshold by adding the background resistance decreased the

amplitude of the load that remained detectable and the RREP was not present for the load

forced below the detection threshold by the elevated Ro. This further supports the

conclusion that the amplitudes of RREP peaks are directly related to the detectability and









magnitude of the load. RREP elicited by inspiratory occlusion was found unaffected by

increased background resistance in the present study. This result was consistent with

previous finding that the RREP elicited by the inspiratory occlusion remains unchanged

with different type of loads and different background environments (Davenport et al.,

1981).

Variations in RREP latencies and amplitudes in the current study provided

evidence that cortical processes to mechanical loads were affected not only by the

magnitude of load but also by the detection of the loaded information, suggesting a gated

respiratory afferent feedback system. In the present study, the P1 peak amplitude of the

RREP is not present with the increased background resistance because the applied

resistive load is below the detection threshold ratio. The fact that the RREP is not present

with increased background resistance suggests that the respiratory resistive load

information was gated out by changing the background ventilatory state, such as

increased background resistance. Thus, the respiratory afferent information will be gated

in if above the detection threshold or will be gated out if the threshold was unregulated by

other systems, such as increased background resistance.

Although the interaction between the conscious and the automatic control of

breathing is uncertain, failure to have sufficient respiratory motor drive to meet the

ventilatory demands will result in a conscious awareness of breathing and behavioral

motor response. Thus, there must be a subcortical threshold to gate-in or gate-out the

respiratory afferent input and feedback motor output. However, neural indicator and the

possible mechanisms that subserve the respiratory sensory gating system are unknown.









Conclusion

In summary, the present study demonstrated that activation of cortical neural

activity was associated with the perceptual processing of resistive load information. The

detection threshold of inspiratory loads was increased by the increased background

resistance but the relative threshold AR50/Ro is unchanged. If the resistive load was

detectable, the RREP was present. If the load was not detectable or becomes undetectable

with increased background load, the RREP was not present, supporting the hypothesis

that the RREP response could be changed by increasing the background load which shifts

the detection threshold. Therefore, perceptual processing of respiratory load information

may be gated-in subcortical centers as part of the central afferent pathway. Future studies

are necessary to investigate the respiratory patterns and the cognitive component of the

RREP P300 peak to extend these studies to the gating effects of the cognitive measures of

respiratory mechanoreceptor processing.









Table 4-1 Group-averaged P1, N1, and Nf latencies and amplitudes of RREP for
inspiratory resistive load and inspiratory occlusion.
P1 C3' P1 C4' N C3' N1 C4' Nf- F3 Nf- F4
Amplitude

R2 No 1.40 + 1.21 + -2.30 + -2.38 + -1.77 -1.71
Background 0.69 0.53 0.85 0.90 0.98 0.62

R3 No 2.20 2.29 -2.85 -3.14 + -2.27 -2.32
Background 0.73# 0.57 #* 1.04 1.41 0.78 #* 0.85 #*
Occlusion 2.31 2.38 -2.02 -2.31 -2.09 -2.11 +
No 0.93 0.97 0.39 0.65 0.80 0.20
Background
R3 With 1.79 + 1.79 + -2.45 -2.54 -1.66 -1.70 +
Background 0.75 0.74 0.88 0.95 0.97 0.81
Occlusion 1.85 + 2.12 -3.01 -3.13 + -1.69 -1.72
With 0.22 0.67 0.93 1.00 0.40 0.32
Background
Latency
(msec)
R2 No 65.73 + 104.74 34.88
Background 7.19 8.99 5.64
R3 No 63.57 + 100.54 36.75
Background 6.70 10.05 5.84
Occlusion 64.53 104.61 34.05
No 4.61 7.50 5.61
Background
R3 With 72.20 109.60 35.30 +
Background 8.40 13.52 3.76
Occlusion 69.98 105.09 31.28
With 9.02 10.24 6.37
Background

Values were means SD. Occlusion was an infinitive load (maximal load) for the
respiratory system.
# indicated the significant difference between R2 and R3 resistive loads.
* indicated the significant difference between no-background resistance and increased
background resistance.











No background resistance


Nf


sec


With background resistance


- R1




R2


sec


Figure 4-1. Nf peaks recorded from F4 electrode with different resistive loads in both no-
background resistance (a) and increased background resistance (b).


(a)







10 li


(b)






10 uV








No background resistance


iw R1


P1


N1


N1
I I I


sec


With background resistance


V


P1


%"' R3


0.8


sec


Figure 4-2. P1 and N1 peaks recorded from C4' electrode with different resistive load in
both no-background resistance (a) and increased background resistance (b).


10 V


10 pV


R1


- R2


I I I I I I I I I I


%OAl


N44










RREP peak response to load
3.5 P1-no load
---. --- P1-with load
....A N1- no load
-A"" ... N1-with load
3 -*- Nf-no load
S...--- Nf-with load

2.5


C 2 2-


1.5


1
load magnitude



Figure 4-3. P1, N1 and Nf peaks recorded from C4' and F4 electrode sites, respectively
with inspiratory occlusion (maximal resistive load) in both no-background
resistance and increased background resistance conditions.



































Figure 4-4. Relationship between amplitude of Pl peak and changes of resistive load
magnitude with different background resistance. The change in resistive load
magnitude was determined by resistive load magnitude divided by baseline
resistance (AR5o/Ro). Ro= 4.8 cmH20/f/s was the baseline resistance
measured from both subject and apparatus with no-background load. Ro'= 16.4
cmH20/f/s is the baseline resistance measured from both subject and
apparatus with increased background load. The ratio (AR5o/Ro) is 0.3 for
normal subject. R2 became undetectable as it was below 0.3 after increasing
the background resistance. Therefore, there was no P peak present when R2
load was applied with increased background resistance.


P1
2.5


2
R2 No-background
a)
1.5
E R3 with-background
E 1



0.5
R2 with-background
0 1I
0.3
0 03 1 2 3

dR/RO














CHAPTER 5
GENERAL DISCUSSION

Summary of Results

1. The results of the evoked potentials elicited by inspiratory occlusion and

cutaneous stimulation demonstrated that short-latency components of the RREP and SEP

project to the same scalp locations. The RREP components were observed bilaterally

whereas the SEP was observed contralateral to the stimulated hand. The scalp

distributions of the RREP and SEP P1 peaks were found at the centroparietal (C4')

electrode position. The latencies of the P1 and N1 peaks of the RREP were significantly

longer than the SEP. The amplitudes of the P1 and N1 peaks of the RREP were

significantly greater than the SEP. The Nf peak was only observed in the frontal region of

RREP.

2. The P1 and N1 peaks of the RREP elicited by inspiratory occlusion were similar

to the peaks of the SEP elicited either by air puff stimulation to the mouth or cutaneous

mechanical stimulation to the chest. The Nf peak was only observed in the frontal sites of

RREP. The latencies of the RREP peaks were longer than the mouth SEP and chest SEP.

The P1 and N1 peak amplitudes of the RREP were significantly greater than the chest

SEP. The N1 peak amplitude of the RREP was significantly greater than the N1 peak of

the mouth SEP. There was no significant difference between chest SEP and mouth SEP

for P1 and N1 peaks.

3. The RREP response to resistive load magnitude was correlated with the presence

of the RREP P1, N1, and Nf peaks with detectable resistive loads. The RREP was not









present with loads that were below the detection threshold. When an increased

background resistance was applied, the RREP peaks were present with the load that was

above the detection threshold but were absent with the load that was below the detection

threshold.

Distribution of Evoked Potentials

RREP with Inspiratory Occlusion

RREP peaks, P1, N1 and Nfwere identified in response to inspiratory occlusion,

consistent with previous studies (Davenport et al., 1996; Davenport et al., 2000;

Davenport et al., 1986). The amplitude of the P1 peak of the RREP increased with the

magnitude estimation of the corresponding load and the inspiratory load magnitude

(Knafelc and Davenport, 1997; 1999). In the present study, these RREP peaks were

recorded in all three experiments. The RREP was not present however, if a load was

below the load detection threshold. The P1, Nf and N1 peaks of the RREP were found

bilaterally. The P1 peak location is consistent with the dipole in the somatosenory cortex.

The Nf peak location is consistent with the dipole in the supplementary motor cortex. The

N1 peak location is consistent with the midline of the cortex, greatest at the vertex.

SEP with Mechanical Stimulation

The P1 and N1 peaks of the limb SEP, mouth SEP and chest SEP were identified in

scalp locations that were contralateral to the side stimulated. These peaks were not

bilaterally symmetrical, a difference from the RREP. The electrode sites of the P1 peak of

the limb SEP, mouth SEP and chest SEP were over the post-central gyms lateral to the

midline which is consistent with the somatotopical representation of the fingers in the

primary somatosensory cortex (Pratt and Starr, 1981). Thus, the mechanical SEP and the

RREP P1 peaks are consistent with an activation of neurons in the primary









somatosensory region of the cerebral cortex. The electrode sites of the N1 peak of the

limb SEP, mouth SEP and chest SEP were over the central sulcus at the midline of the

cerebral cortex which is also consistent with the somatotopical representation of the

fingers in the sensorimotor cortex (Pratt and Starr, 1981). Thus, the mechanical SEP and

the RREP N1 peaks are consistent with an activation of neurons in the sensorimotor

region of the cerebral cortex.

Mechanical stimulation activates mechanoreceptors in a defined focal location in

the hand, chest or buccal surface of the mouth. The mechanoreceptors are myelinated

afferents with a rapid conduction velocity. The latency for mechanical cutaneous

stimulation included mechanical transduction time and pathway conduction time. The

latency for skin (limb and chest) mechanoreceptors to the cortex is less than either the

mouth or RREP. However, the cutaneous peak amplitudes were smaller, most likely due

to fewer mechanoreceptors activated by the focal stimulation compared to the widely

distributed stimulation with inspiratory occlusion.

Latency and Amplitude of Short-Latency Peaks of Evoked Potential

RREP

The P1 peak of the RREP has been reported to reflect the arrival of respiratory

mechanoreceptor activity in the primary sensory area of the cortex (Logie et al., 1998).

The P1 peak is determined mainly by the physical characteristics of the stimulus. The

RREP P1 peak amplitude was correlated to the resistive load magnitude (Knafelc and

Davenport, 1997; Webster and Colrain, 2000), suggesting that this peak is the indicator of

the stimulus dependent activation of somatosensory cortical columns. The amplitude of

RREP N1 peak was reported to decrease with a decrease of the load magnitude (Bloch-

Salisbury et al., 1998). The P1 and Nf peak amplitudes were unaffected by attention









while attention increased the N1 amplitude (Harver et al., 1995). Zhao et al. (2002) also

reported that there were no significant difference in either latency or amplitude of the P1

and Nf peaks of the RREP peaks between attend trials and ignore trials for both double

lung transplant recipients and matched healthy subjects. Thus, the P1 and Nf peaks are

dipoles in the cerebral cortex indicating the arrival and magnitude of respiratory afferent

activation.

It is unlikely that the frontal Nf peak of the RREP reflects myogenic activity. The

neural generators of the Nf peak are consistent with a pre-motor cortical location. The

bilateral distribution of the Nf peak and the latency suggest that it reflects a parallel (to

P1) input to activity in prefrontal structures. It is generally recognized that

thalamocortical inputs to the somatosensory cortex project predominantly to deep cortical

layers and that they generate a surface (P1) positive potential (Wood and Allison, 1981;

Wood et al., 1985). The frontal Nf peak was not preceded by the P1 peak and can not be

explained as voltage spread from the somatosensory cortex. The negative peak suggest a

dipole generator in the more superficial layers of the prefrontal cortex

The prefrontal cortex gated the early stage of somatosensory processing and can

exert an inhibitory modulation on sensory processing (Yamaguchi and Knight, 1990).

The prefrontal cortex mediates cortical motor reflexes suggesting that the generation of

the frontal Nf potential is a pre-motor respiratory cortical response to the respiratory load.

The Nf peak generated in the prefrontal cortex may therefore function as the integration

of load information related to the behavioral motor response to the mechanical stimulus

produced by loaded breathing (Davenport et al., 1996). However, the exactly source of

the Nf peak is unknown.









The N1 peak of the RREP was the negative peak associated with the P1 peak of

the RREP (Davenport et al., 1986). The N1 peak of the RREP was observed to be of

greatest amplitude in the central electrode sites. These electrode sites were over the

central sulcus at the midline of the cerebral cortex which is consistent with the

somatotopical representation of the fingers in the sensorimotor cortex (Pratt and Starr,

1981). In addition, Webster and Colrain (2000) reported N1 peak is recorded from the

vertical dipole source and is affected by the attention. Thus, the N1 peak of RREP may be

the second order cortical processing of respiratory afferent information. Bloch-Salisbury

et al. (1998) reported that N1 peak amplitude of the RREP decreases as the resistive load

decreased, similar to P1 peak of the RREP. Thus, the N1 peak of the RREP may be the

second order cortical processing of the respiratory afferent information that is based on

perceiving the first-order somatosensory information and may affect the cognitive

processing.

SEP on Hand

The P1 peak was elicited by mechanical cutaneous stimulation of the load in the

C3' and C4' electrode sites in the present study. This is consistent with the reports of SEP

elicited by mechanical stimulation of the digits (Onofrj et al, 1990; Pratt and Starr, 1981),

suggesting that the generation of this early positive peak was in the primary

somatosensory region of the cortex (Hamalainen et al., 1990). In the current study, the

latency of the SEP P1 peak elicited by cutaneous stimulation of the hand was about 25

msec. This is consistent with the report of Schieppati and Ducati (1984) that the P1 peak

of the SEP is elicited by the cutaneous stimulation. Thus, the results of the present study

are consistent with the P1 peak of the SEP generated in the somatosensory region of the

cortex.




Full Text

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RESPIRATORY MECHANORECEPTOR ACTIVATION OF SOMATOSENSORY CORTEX IN HUMANS By YANGLING CHOU A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by Yang-Ling Chou

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This dissertation is dedicated to my beau tiful homeland, Taiwan, and to my dearest parents, Dr. I-Chang Jou and Pao-Chu Huang

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ACKNOWLEDGEMENT First and foremost, I would like to express my gratitude toward my mentor, Dr. Paul W. Davenport, for having me as his graduate student. His enthusiastic support and great guidance on many aspects of this dissertation were invaluable. I also gratefully acknowledge helpful support from the rest of my committee, Dr. Daniel Martin, Dr. Linda Hayward, Dr. Donald Bolser and Dr. Christine Sapienza, for consistently providing me with valuable comments and suggestions during my graduate work. My special thanks will go to Kan-Nian Hu, my very significant other whom I met during this graduate work. His warm and thoughtful support was always my driving force towards this graduate work. I would like to thank my father, Dr. I-Chang Jou; my mother, Pao-Chu Huang; my two sisters, Lin-Ru Chou and Yang-San Chou, for their encouragement and support during my graduate work. My thanks also go to my colleagues for their thoughtful help. They are Dr. Weirong Zhang and Camille Swartz. Their involvement was very helpful towards my graduate work. I would also like to thank some friends for their support. They are Keng-Liang Huang, Hsiao-Lan Sun, Dr. Chien-Hui Huang, Yuming Guo and Jeau-Jun Chen. I always enjoy the happiness and talks we have shared together. Finally, very special thanks go also towards all the subjects who participated in my studies and contributed to my dissertation work. It is their cooperativeness and patience that made such successful dissertation work. iv

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TABLE OF CONTENTS page ACKNOWLEDGEMENT.................................................................................................iv LIST OF TABLES...........................................................................................................viii LIST OF FIGURES...........................................................................................................ix ABSTRACT.......................................................................................................................xi CHAPTER 1 REVIEW OF CORTICAL PROJECTION OF RESPIRATORY MECHANORECEPTORS ACTIVATION IN RESPONSE TO MECHANICAL LOAD...........................................................................................................................1 Cortical Projection of Respiratory Mechanoreceptors.................................................1 Respiratory Related Evoked Potential..........................................................................2 Potential Sources and Afferent Mechanisms of RREP.................................................6 Mechanical SEP and Somatosensory Pathway...........................................................11 Perception of Mechanical Loads................................................................................14 Summary of Experiments...........................................................................................18 Rationale for Study 1...........................................................................................19 Rationale for Study 2...........................................................................................20 Rationale for Study 3...........................................................................................22 Summary of Hypotheses.............................................................................................23 2 THE CORTICAL RESPONSES TO INSPIRATORY LOADS AND TACTILE STIMULATION FOR HEALTHY ADULTS............................................................24 Introduction.................................................................................................................24 Methods......................................................................................................................28 Subjects................................................................................................................28 Pulmonary Function Testing...............................................................................28 RREP...................................................................................................................29 SEP......................................................................................................................30 Statistical Analysis..............................................................................................31 Result..........................................................................................................................31 Discussion...................................................................................................................32 Conclusion..................................................................................................................36 v

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3 CORTICAL EVOKED POTENTIALS ELICITED BY INSPIRATORY OCCLUSION, AIR-PUFF, AND CUTANEOUS STIMULATION IN HEALTHY ADULTS.....................................................................................................................45 Introduction.................................................................................................................45 Method........................................................................................................................47 Subjects................................................................................................................47 Pulmonary Function Testing...............................................................................47 RREP Elicited by Inspiratory Load.....................................................................48 Mechanical Stimulation of the Chest and the Mouth..........................................49 Statistical Analysis..............................................................................................51 Result..........................................................................................................................51 Discussion...................................................................................................................52 Conclusion..................................................................................................................57 4 RESPIRATORY LOAD PERCEPTION AND RREP WITH INCREASED BACKGROUND RESISTANCE...............................................................................66 Introduction.................................................................................................................66 Method........................................................................................................................69 Subject.................................................................................................................69 Pulmonary Function Testing...............................................................................70 Procedures...........................................................................................................70 Data Analysis.......................................................................................................72 Results.........................................................................................................................73 Discussion...................................................................................................................74 Conclusion..................................................................................................................78 5 GENERAL DISCUSSION.........................................................................................84 Summary of Results....................................................................................................84 Distribution of Evoked Potentials...............................................................................85 RREP with Inspiratory Occlusion.......................................................................85 SEP with Mechanical Stimulation.......................................................................85 Latency And Amplitude Of Short-Latency Peaks Of Evoked Potential....................86 RREP...................................................................................................................86 SEP on Hand........................................................................................................88 SEP on Chest and Mouth.....................................................................................89 Afferent Mechanisms Of Evoked Potentials..............................................................90 Sources of RREP.................................................................................................90 Afferent Pathways of RREP and SEP.................................................................91 Comparisons Between RREP and SEP...............................................................92 The Relationship Between RREP and Load Perceptual Process................................93 Relationship Between RREP and Load Perception.............................................93 RREP Responses to Different Load Magnitude..................................................94 Short-Latency RREP Peak in Response to Increased Background Resistance...94 vi

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6 CONCLUSION...........................................................................................................99 LIST OF REFERENCES.................................................................................................101 BIOGRAPHICAL SKETCH...........................................................................................112 vii

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LIST OF TABLES Table page 2-1 Mean amplitude and latency data for the respiratory-related evoked potential and the somatosensory evoked potential..................................................................38 3-1 Group mean amplitudes and latencies for P1 and N1 peaks of the RREP, mouth SEP, and chest SEP..................................................................................................59 4-1 Group-averaged P1, N1, and Nf latencies and amplitudes of RREP for inspiratory resistive load and inspiratory occlusion.................................................79 viii

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LIST OF FIGURES Figure page 2-1 Respiratory-related evoked potential Schmidt.........................................................39 2-2 SEP at C4' and C3' electrode sites (C4'Right hemisphere; C3'-Left hemisphere).40 2-3 RREP at C4' and C3' electrode sites (C4'-Right hemisphere; C3'-Left hemisphere).41 2-4 The RREP and SEP at F4 electrode site. The Nf peak was only observed in the RREP but not in the SEP..........................................................................................42 2-5 Comparison for the amplitudes of the P1 and N1 peaks of the RREP and the SEP...........................................................................................................................43 2-6 Comparison between the C3' and C4' electrode sites for the P1 and N1 peaks of the RREP and the SEP.............................................................................................44 3-1 Comparison of the RREP, Mouth SEP, and Chest SEP from frontal electrode (F4). The black line represents the stimulus.....................................................................60 3-2 Comparison of the RREP from right (C3') and left (C4') electrodes. The black line represents the inspiratory occlusion..................................................................61 3-3 Comparison of the mouth SEP from right (C3') and left (C4') electrodes...............62 3-4 Comparison of the chest SEP from right (C3') and left (C4') electrodes.................63 3-5 Comparison for the amplitudes of the P1 and N1 peaks of the RREP, mouth SEP and chest SEP...........................................................................................................64 3-6 Comparisons between the C3' and C4' electrode sites for the P1 and N1 peaks of the mouth SEP and chest SEP..................................................................................65 4-1 Nf peaks recorded from F4 electrode with different resistive loads in both no-background resistance (a) and increased background resistance (b)........................80 4-2 P1 and N1 peaks recorded from C4' electrode with different resistive load in both no-background resistance (a) and increased background resistance (b)..........81 ix

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4-3 P1, N1 and Nf peaks recorded from C4' and F4 electrode sites, respectively with inspiratory occlusion (maximal resistive load) in both no-background resistance and increased background resistance conditions......................................................82 4-4 Relationship between amplitude of P1 peak and changes of resistive load magnitude with different background resistance.....................................................83 5-1 Proposed respiratory gating model...........................................................................98 x

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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 RESPIRATORY MECHANORECEPTOR ACTIVATION OF SOMATOSENSORY CORTEX IN HUMANS By Yang-Ling Chou May 2005 Chairman: Paul W. Davenport Major Department: Veterinary Medicine Humans can consciously perceive difficulty with their breathing. It is known that humans can sense external respiratory loads by peripheral mechanoreceptors and process the afferent information via several afferent pathways. However, neural pathways processing the conscious sensation of respiratory loads and how this respiratory afferent information projects to higher brain centers are still poorly understood. The respiratory-related potential (RREP) elicited by inspiratory occlusion in humans recorded from the scalp provided a unique way to investigate the neural mechanisms mediating respiratory load information and the activation of mechanoreceptor related brain activity. The major goal of this dissertation was to investigate the activation of respiratory mechanoreceptor eliciting RREP responses to inspiratory loads and its relation to somatosensory cortical activation. Using somatosensory evoked potentials as a model of RREP processing, RREP responses to xi

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inspiratory occlusion and during breathing against inspiratory resistive loads with increased background resistance were studied in this dissertation. The short-latency RREP components were compared with the components of the somatosensory evoked potential (SEP) elicited by mechanical stimulation of the hand, chest, and mouth. Short-latency components of the RREP were also compared between no-background resistance and increased background resistance to determine the mechanism of respiratory baseline state on afferents projecting to the cortex. Three studies were conducted and a total of 60 healthy adults participated. Results from the first two studies demonstrated that the RREP and SEP are both observed over the somatosensory cortex. Unlike the SEP, the RREP appeared to be elicited by afferents that project simultaneously to both sides of the somatosensory cortex. The RREP may have greater amplitude due to greater population of afferents activated with a global stimulus such as inspiratory occlusion. These results further demonstrated that the RREP Nf peak is a unique frontal cortical activation elicited by inspiratory occlusion. The results from the third study demonstrated the RREP can be changed by increasing the background load and shifting the detection threshold of the subject. If the resistive load is detectable, the RREP was present; if the load is not detectable or became undetectable with increased background load, the RREP was absent. xii

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CHAPTER 1 REVIEW OF CORTICAL PROJECTION OF RESPIRATORY MECHANORECEPTORS ACTIVATION IN RESPONSE TO MECHANICAL LOAD Cortical Projection of Respiratory Mechanoreceptors Respiration is regulated by brainstem respiratory centers, which act as intrinsic controllers to generate the respiratory activity. This respiratory activity can be modified by the afferent feedback from both chemoreceptors and mechanoreceptors to meet the respiratory demands. The automatic regulation of breathing can also be overridden by the cognitive centers of forebrain and by conscious control of respiration. Thus, respiration is regulated not only by brainstem automatic controller but also by cortical voluntary motor systems (American Thoracic Society, 1999). Regular breathing can be modified by consciously perceived sensory inputs from changes in ventilation, such as volume, airflow, pressure, or force. It is well documented in human studies that sensory afferents project to the somatosensory cortex (Kakigi and Shibasaki, 1984). Thus, respiratory sensation and perception may result from the cortical integration of respiratory afferent information. Previous studies have shown the projections of low-threshold afferents from intercostals muscles to the cerebral cortex (Gandevia and Macefield, 1989), and phrenic afferents to the somatosensory cortex in both cats (Balkowice et al., 1995) and humans (Zifko et al., 1995; 1996). Cortical evoked potentials elicited by inspiratory occlusion (Davenport et al., 1986; Revelette and Davenport, 1990), direct vagal stimulation (Tougas et al., 1993), and pressure pulse stimulation to the mouth (Daubenspeck et al., 2000) have been reported. These previous 1

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2 works suggested that humans can consciously perceive external added mechanical loads and lung volume changes. This conscious awareness of mechanical load implies that there must be activation of neurons in cerebral cortex that is measurable by electrophysiological approaches. Balzamo et al. (1995) demonstrated that the load estimation of mechanical stimuli applied to the middle finger was significantly lowered by the increased background respiratory resistance in healthy humans. Activation of respiratory afferents by resistive loads also modified the somatosensory evoked potentials elicited by median nerve stimulation in humans (Balzamo et al., 1999). Thus, respiratory afferents activated by mechanical loads may have similar pathways as other somatosensory modalities and may project to the somatosensory cortex. If respiratory mechanical afferents are processed in part by the somatosensory cortex, then neurons of the somatosensory cortex must be activated by respiratory mechanoreceptors. In animals, afferent information processed from both phrenic afferents and intercostals muscle afferents has been demonstrated to activate the thalamus (Zhang and Davenport, 2003) similar to mechanosensation in other sensory modalities (Yates et al., 1994). In addition, phrenic afferents and intercostals muscle mechanoreceptors have been demonstrated to activate neurons of the somatosensory cortex in cats (Davenport et al., 1985; Davenport et al., 1993). Thus, respiratory mechanical afferents may project to the somatosensory cortex via the pathway that is similar to other sensory modalities. Respiratory Related Evoked Potential Evoked potentials have been used to record the cortical neural activity elicited by a synchronous stimulation of afferents that project to specific regions of the cortex. The respiratory-related evoked potential (RREP) elicited by inspiratory occlusion in humans

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3 was recorded over the somatosensory cortex (Davenport et al., 1986). The RREP was present bilaterally in normal healthy subjects (Revelette and Davenport, 1990). The latencies of the RREP were inversely related to the respiratory drive and the increases in RREP amplitude were related to the magnitude of the inspiratory load (Knafelc and Davenport, 1997). In addition, the RREP was identified bilaterally in all subjects and the greatest amplitude of the P1 peak of the RREP was found in somatosensory cortex (Davenport et al., 1986; Logie et al., 1998). The RREP was reported using negative pressure mechanical stimuli, expiratory occlusions (Strobel and Daubenspeck, 1993; Hammond and Davenport., 1999) and different timing of inspiratory stimuli (Davenport and Revelette, 1990). Measurement of the RREP also provided a unique way to investigate the cortical activities elicited by respiratory loaded breathing. Humans can consciously perceive changes of airway pressure (Younes and Sanii, 1989). RREPs were recordings that reflected the cortical activities in response to inspiratory occlusion and inspiratory mechanical obstruction. Thus, it is reasonable to expect that physiological stimulation should produce a RREP with components reflective of the nature of afferent activation of respiratory mechanoreceptors distributed throughout the respiratory system. However, the sources of mechanoreceptors and afferent mechanisms mediating the RREP are unknown. The RREP elicited by inspiratory occlusion has been reported in animals (Davenport et al., 1985; Davenport et al., 1991; Davenport et al., 2001), children (Davenport et al., 2000), and adult subjects (Davenport et al., 1986; Revelette and Davenport, 1990). A consistent pattern of three components of the RREP (Nf, P1, N1) has been identified in response to occlusions presented either at the onset of inspiration or

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4 by the mid-inspiration (Davenport et al., 1986; Davenport et al., 2000; Davenport et al., 1996). The greatest amplitude of the P1 peak of the RREP has been identified by using dipole source techniques demonstrates that the generator of the P1 was bilaterally located within primary somatosensory (Davenport et al., 1986; Logie et al., 1998). The P1 component of the evoked potential has been demonstrated to reflect the integrity of primary sensory pathway and varies in morphology with stimulus parameters such as intensity and frequency (Strautus et al., 1997). Results from RREP studies using different levels of resistive loads suggested that the P1 peak is a cortical marker of the arrival of the mechanical sensory signal to the sensory cortex (Campbell et al., 1961; Johnson, 1993; Davenport et al., 1986). These findings suggested that the somatosensory cortex is one part of neural circuitry sub-serving respiratory load perception. In addition, Franzn and Offenloch (1969) reported that the amplitude of SEP components was correlated with the magnitude estimation of the stimuli. Similar results have been demonstrated in the RREP study that the amplitude of RREP P1 peak increased as the increase of resistive load magnitude (Knafelc and Davenport, 1997). The N1 peak was found in the electrodes over the vertex, Cz (Davenport et al., 1986). The N1 peak can be recorded from the vertical dipole source and is affected by attention (Webster and Colrain, 2000). Evidence for cortical neural processing of respiratory sensation has been identified by using the dipole modeling and concluded the generators of N1 peak were bilaterally located within supplementary motor cortex (Logie et al., 1998). Thus, the N1 peak may be related to the second-order afferent information

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5 processing to the load (Davenport et al., 1996) and may be related to attention (Webster and Colrain, 2000). A unique negative peak, Nf, has been found at the pre-central electrodes 25-40 ms after the occlusion (Davenport et al., 1996). This short latency frontal peak (Nf) of the RREP represents respiratory load information processing which may be parallel to the somatosensory P1 peak information. The Nf-P1 was hypothesized to represent parallel activation of somatosensory cortex and prefrontal cortex (Logie et al., 1998).The short latency Nf peak has only been found in the pre-central electrodes referenced to the joined earlobes and the P1 peak has not been recorded from frontal electrodes (Davenport et al., 1986), suggesting that dual cortical generators separate via thalamic pathways (Hamalaimen et al., 1990). However, the exact neural mechanism of the Nf peak remains unknown. The long-latency components of the RREP reflect cognitive and psychological states of the subject. They are influenced primarily by factors extrinsic to the physical stimulus, such as arousal, attention, motivation, and task difficulty and are thought to be related to the cognitive processing of respiratory load information (Bloch-Salisbury et al., 1998; Colrain et al., 1996; Harver et al., 1995; Webster and Colrain, 2000). The RREP P300 peak was found to exhibit changes in amplitude and latency in response to cognitive processing of the stimulus information and perceptual factors (Bloch-Salisbury et al., 1998; Webster and Colrain, 2000). The role of the short-latency components of the RREP and their relationship with other mechnosensory modalities remains unknown. Therefore, a series experiments has been designed in the current dissertation to investigate the latencies and amplitudes of

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6 short-latency peaks of RREP that are indicators for cortical processing of respiratory load information. Potential Sources and Afferent Mechanisms of RREP Afferents in many structures of the respiratory system may contribute to the RREP and the RREP may be the summation of activation of several afferent populations. Respiration against mechanical loads can alter the afferent activity of mechanoreceptors in the mouth, pharynx, larynx, lung and respiratory muscles. The inspiratory occlusion elicits the RREP reflecting the activation of the mechanoreceptors distributed throughout the entire respiratory system (Davenport et al., 1986).The afferent information from these mechanoreceptors is mediated via the facial nerve, vagus nerve, superior laryngeal nerve, recurrent laryngeal nerve, phrenic nerve, and other respiratory muscle nerves. Activation of cortical neurons by these afferents has been reported (Davenport et al., 1986; Zifko et al., 1996; Togus et al., 1993; Gandevia and Macefield, 1989). However, the afferents mediating inspiratory load information and the neural process associated with loaded breath that elicits the RREP remain uncertain. The activation of cortical neural activity by respiratory mechanical loads has been studied using evoked potential techniques similar to those used in recording the SEP. The fact that subjects are consciously aware of breathing against the mechanical loads suggests that there must be activation of neurons in the cerebral cortex. Chou and Davenport (2004) have demonstrated that stimulation of phrenic afferents would elicit cord dorsum potential in the dorsal horn of the cats spinal cord. In this study, the phrenic afferents activated by single pulse stimuli were primary group I and II, and to a lesser extent group III afferents. Electrical stimulation of phrenic afferents has been shown to elicit evoked potential in the area 3a and area 3b of the somatosensory

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7 cortex (Davenport et al., 1985). In addition, cortical evoked potentials elicited by both electrical and mechanical stimuli have been reported (Davenport et al., 1993). This study reported that intercostal muscle afferents project to the area 3a of the somatosensory cortex. Stimulation of phrenic afferents activates the neurons in the ventroposterior lateral nuclei (VPL) of the thalamus (Davenport and Reep, 1995). The stimulation of intercostals muscle afferents activated different neurons in the VPL of the thalamus (Davenport and Revelette, 1995). Cortical evoked potentials found in the somatosensory cortex were associated with C5 root stimulation (Yates et al, 1994). Retrograde fluorescent tracers in this study found in labeled cells in the oralis nuclei of the ventroposterior complex (VPO) of the thalamus of cats suggested that phrenic afferents project to the somatosensory cortex via thalamocortical pathway. Straus et al. (1997) reported cortical evoked potentials elicited by phrenic stimulation in humans. In addition, Gandevia and Macefield (1989) demonstrated cortical projections by stimulating intercostals muscle afferents in humans. Evidence of neural substrates mediating the afferent information from respiratory mechanoreceptors to the somatosensory cortex has thus been provided from both animal and human studies. Therefore, information related to respiratory mechanical activity may be transduced by respiratory mechanoreceptors and project to the somatosensory region of the cortical cortex via a dorsal column lemniscal pathway, which is relayed at the VPL and/or VPO of the thalamus. Single pulse vagal nerve stimulation elicited activity in sensory cortex and thalamus in the monkey (Hallowitz and MacLean, 1977). A similar study in cats reported that cortical evoked potentials recorded in both the somatosensory cortex and motor

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8 cortex elicited by vagal afferents (Korn and Massion, 1964). A positive peak of the evoked potential with a latency of about 21-22ms was elicited by natural cutaneous stimulation directed to the skin of the face (Schieppati and Ducati, 1984). Similarly, two subsequent studies also showed that both facial and trigeminal nerve afferents could elicit neural activity in the somatosensory cortex in human (Hashimoto, 1987; Seyal and Browne, 1989). Cortical activities elicited by superior laryngeal nerve stimulation have also been recorded in cruciate sulcus, anterior sylvian sulcus, suprasylvian sulcus and orbital cortex in cats (Aubert and Guilhen, 1971). There are two potential afferent pathways for lung vagal afferents: one projects to the somatosensory and motor cortices and a second projects to the mesocortex. Although there is limited information concerning vagal afferent pathways to the somatosensory cortex and the neural activity elicited by vagal afferent activation in humans, mechanoreceptor information from the upper airway and lung has been demonstrated to elicit cortical activation in response to negative pressure (Daubenspeck et al., 2000). The presence of both earlyand lateRREP peaks in double lung transplant recipients with and without tracheostomy suggested lung vagal afferents and upper airway mechanoreceptors elicit a RREP in response to inspiratory occlusion but are not essential sources of the RREP response (Davenport et al., 2004; Zhao et al., 2002; 2003). Thus, respiratory related afferents activated by inspiratory occlusion or negative pressure pulses elicited cortical neural activity. This information may arise from respiratory muscles, the lung and/or the upper airways. Breathing against an external resistive load changes the pressure, volume, and airflow in the entire respiratory tract. These mechanical changes are mediated by

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9 mechanoreceptors from the lung, upper airway and respiratory muscles. There are many potential afferent sources and pathways that contribute to respiratory perception of loaded breaths. However, it is difficult to determine the specific neural mechanism subserving load related afferent information processing because the load dependent mechanical events are transmitted throughout the entire respiratory tract. Several sensory sources may contribute to respiratory sensation. Among them, respiratory muscle afferents probably play a primary role. However, the roles of receptors in the upper airway and lung remain equivocal. Application of external loads to the airway increases the transluminal pressure gradient across the extrathoracic airways. This changes the airway wall configuration and consequently modulates the sensory information arising from those upper airways. Both animals (Davenport et al., 1985; Davenport et al., 1993; Davenport and Hutchison, 2001) and humans (Davenport et al., 1986; Davenport et al., 1996; Knafelc and Davenport, 1997) have been studied to understand the afferent mechanism of the RREP. Animal studies of respiratory sensation have been developed in dogs with tracheal stoma. These dogs were behaviorally conditioned to detect inspiratory resistive loads and occlusions. The stoma in these dogs excluded afferent information from the upper airway mechanoreceptors. It was found that dogs with a tracheal stoma could reliably detect inspiratory resistive loads and occlusion (Davenport et al., 1991). Thus, it appears that afferents in the mouth, nose, pharynx, larynx and upper extrathoracic trachea are not mediating load detection in these dogs. The remaining intact sources diaphragm muscle mechanoreceptors and intercostal muscle mechanoreceptors were likely the afferents mediating the load information. Phrenic and intercostals muscle afferents have been reported to activate neurons in somatosensory

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10 region of the cat brain (Davenport and Reep, 1995; Davenport and Revelette, 1995). Animal studies of the neural mechanism mediating respiratory sensations have demonstrated that respiratory muscle afferents may be, in part, mediating respiratory sensation (Davenport et al, 1985; Davenport et al., 1993; Davenport and Hutchison, 2001; Zhang et al., 2003). The occlusion-elicited evoked potential, which is analogous to the RREP in human studies, has been recorded in the somatosensory cortex of awake lambs (Davenport et al., 2001). The RREP was present when the lambs respired via their mouth and via an endotracheal tube. Stimulation of intercostal muscles (Gandevia and Macefield, 1989), vagal afferents (Tougas et al., 1993), supraglottal afferents (Daubenspeck et al., 2000), and phrenic afferents (Straus et al., 1997; Zifko et al., 1996) activates the somatosensory cortex with short latency evoked potentials in humans. This somatosensory cortical projection is via the thalamus which is similar for mechanoreceptor sensation for other sensory systems. However, the neural mechanism and afferent pathway of the RREP elicited by inspiratory occlusion in humans remain unknown. In summary, there may be two cortical pathways involved in respiratory central neural processing (Davenport et al., 1995). One is the pathway for respiratory muscle afferents. Neural information arising from respiratory mechanoreceptor enters the spinal cord, ascends in the dorsal column, relays in the brainstem, projects to the ventroposteral region in the thalamus via the medial lemniscal tract, and is projected through a thalamocortical pathway to the sensorimotor cortex (Zhang et al., 2003). The function of this pathway could relate to the mechanical events of respiratory muscles. The second pathway involves ascending afferent information from pulmonary stretch receptors via

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11 the vagal nerve and its branches, and possibly the upper airways mediated by the RLN, SLN, trigeminal and facial nerves. They relay sensory information to the brainstem and then project to the mesocortex. This circuit may be related to the behavioral aspect of respiration. Mechanical SEP and Somatosensory Pathway There are receptors that inform the brain about mechanical stimuli from touch, pressure, and vibration (Mountcastle, 1980). Cortical evoked potentials can be elicited by different sensory modalities (Mima, 1997). Evoked potentials have been studied extensively in humans from somatosensory modalities and provide information regarding cutaneous potential fields generated by synchronous activation groups of neurons. In addition, evoked potentials generated in the cerebral cortex have an inversion of polarity as the electrode is lowered from the surface to underlying cortex; that is, a change of polarity is recorded as an electrode passes from the positive voltage region (source current) to the negative voltage region (sink current). An inversion of polarity of large amplitude focal potentials thus provides strong evidence for the presence of a local generator with dipolar properties (Wood and Allison, 1981). The activation of somatosensory neuronal activities has been studied and well defined for its onset latency by recording the sensory evoked potential (SEP) associated with electrical and mechanical stimulation. Onofrj et al. (1990) concluded that short-latency SEP components recorded following mechanical stimuli were of longer latency and smaller amplitude than evoked potentials recorded following electrical stimuli. This result was supported by the findings from Buchthal (1980) that more time was needed for skin indentation and the process of receptor transduction with mechanical stimuli as compared to electrical stimuli.

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12 The SEP is the result of the summed activity in the somatosensory cortex in response to somatosensory afferent input. The SEP can be expected with physiological stimuli and can be used as a method for measuring electrophysiological and perceptual events. Cortical surface recordings show that the early peaks of SEP were evoked only by contralateral stimulation; ipsilateral stimulation did not produce detectable activities (Lder et al., 1986; Wood et al, 1988; Allison et al., 1989). Studies of the SEP have focused on the sources and components of the SEP considered to reflect both cortical and subcortical activation of the brain (Desmedt et al., 1983, 1984; Kekoni et al., 1997). In fact, the early components of the SEP following mechanical stimulation have been found to correlate with stimulus magnitude and magnitude estimation and are a summation of spatially and temporally overlapping potentials that may have separate origins (Hashimoto et al., 1987). Since the SEP is the result of summed activity in the somatosensory cortex in response to afferent outputs, methods can be used to quantify both electrophysiological and perceptual events (Regan et al., 1984). The short-latency component of the SEP elicited by mechanical stimulation was correlated with the magnitude of the stimulus and the magnitude estimation of the subjects (Franzn and Offenloch, 1969; Hashimoto et al., 1992). The spatiotemporal pattern of these recordings has been demonstrated in previous studies that a series of two early peaks recorded within 100 ms after stimulation were largest over the contralateral somatosensory cortex (Kakigi and Shibasaki, 1984; Hashimoto, 1987). Inputs from the somatosensory cortex were essential for activation of other cortical regions. This may explain why lesions or surgical removal of the hand area of the human somatosensory cortex appears to abolish all cortical SEP peaks (Stohr and Goldring, 1969; Williamson et al., 1970; Allison et al.,

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13 1992). Patients with cerebral lesions producing abolition of contralateral short-latency SEP also exhibit bilateral loss of long latency SEP peaks (Williamson et al., 1970). The short-latency peaks of the SEP originate from the somatosensory cortex located in the rostral bank of post central gyrus (Allison et al., 1989a; Allison et al., 1991; Wood et al., 1988) in humans and indicate that cutaneous mechanosensation is initially processed in somatosensory regions of the cortex (Geyer et al., 1999). Results from direct subdural strip recordings have shown that the occurrence of the early peaks in the somatosensory cortex appears to play a necessary but not sufficient role in conscious awareness of an afferent stimulus (Ray et al., 1999). Peterson et al. (1995) reported in monkeys that short-latency components of epidural SEP elicited by median nerve stimulation were the result of separate generators, which consisted of a number of dipole sources in precentral gyrus (frontal electrodes). Information related to different sensory modalities is initially transmitted along parallel, modality-specific, multisynaptic pathways (Mountcastle, 1980). Information detected by external sensory receptors is relayed via the pathway stations in the brainstem to thalamus, then projected to specialized areas of the cortex. Studies of primates have demonstrated that the primary somatosensory cortex has not only a somatotopic but also modality-specific organization (Kaas, 1991). Area 3b and area 1 were mainly activated by cutaneous stimulation, while area 3a receives afferent input from deep sensory receptors and area 2 plays a somatosensory integrative function for all modalities. Previous studies have shown the projections of intercostal muscle afferents and of the phrenic afferents to the somatosensory cortex (Gandevia and Macefield, 1989; Davenport et al., 1993; Zifko et al., 1995; 1996). These results support the conclusion that cortical

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14 activation was the summation of respiratory afferents projecting to the sensory region of the cortex. Perception of Mechanical Loads Mechanoreceptors activated by mechanical loads during respiration and the pathway mediating the afferent information to higher brain center play an important role for the perception of respiratory obstruction. Perception of respiratory mechanical events depends on cognitive processing of afferent information from peripheral mechanoreceptors. This is measured in conscious humans by determination of the threshold value of a stimulus required for detection and/or the estimation of load magnitudes over a range of stimuli above the detection threshold. Signal detection may only require activation of a set of parallel somatosensory pathways, but magnitude estimation involves cognitive evaluation. Sensation of respiratory resistive loads, using psychophysical studies of load detection and magnitude estimation of respiratory resistive load, is similar to other mechanical load interventions to the respiratory system (Killian et al., 1980; Stubbing et al., 1981). Psychophysical methods have been used to quantify respiratory sensation (Wiley and Zechman, 1966). The perception of respiratory mechanical events, such as an applied resistive load, is dependent on two processes (Johnson, 1993). The first step is load detection, which has been studied by using difference threshold methods (Campbell et al., 1961; Davenport et al., 1986). The second perceptual process includes differentiation of the load type and estimation of the load magnitude. Experimentally, subjects were required to provide an estimate of the sensory magnitude of a load above the detection threshold using a numerical scale or cross-modality matching, such as handgrip tension sensation to match the magnitude of respiratory sensation (Davenport et

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15 al., 1996; Revelette and Davenport, 1990; Davenport et al., 1991). These studies showed that the perceived magnitude of an increased extrinsic load is linearly related to the load magnitude when a log-log transformation is used. The slope of the line is a measure of the sensitivity of the subject to the stimulus. Adult humans can perceive and scale respiratory mechanical loads (Bennett et al., 1962; Buki et al., 1983; Strobel et al., 1993; Fitzpatrick et al., 1995). The mechanical loads are added to the breathing pathway of human subjects. Resistive loads alter respiratory pressure-flow dynamics. Added resistive loads are constructed of porous material and are connected in series to a breathing circuit to provide linear increases in airflow resistance. Resistive loads serve to mimic the type of internal airflow limitation experienced by patients with obstructive lung disease such as asthma. Typically, a series of known resistive loads are added for one or several breaths and the subject indicates if the added load is sensed (Zechman et al., 1979). The detection of respiratory loads is dependent on the background resistance of the subjects airways and the breathing apparatus (Wiley and Zechman, 1966). The ability of a subject to detect an added load, R 50 is a function of the ratio of the added load to the background load (R 0 ) resulting in the Weber-Fechner Ratio, R 50 /R 0 The R 50 /R 0 for normal subjects is 0.3, meaning that the R 50 must be 30% above the total R 0 for the load to be detected. This R 50 /R 0 is also 0.3 for COPD patients with elevated background resistance. The detection threshold R 50 is higher as is the R 0 but the ratio remains constant. Ventilation is a mechanical process with respiratory muscles generating the driving force to the respiratory pump. Breathing against an external mechanical load changes the breathing pattern in a load-specific manner. Inspiration against a suprathreshold load

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16 results in a reduction of airflow for a resistive load (Zechman and Davenport, 1978). Inspiratory obstruction alters the mechanical breathing process and leads to a conscious perception of the mechanical loads. The pattern of airflow, volume and pressure changed by adding the resistive load and the perception of the added resistive load is also changed by adding a background load (Revelette et al., 1990). Perceiving a loaded breath is affected by the increased background resistance. The detection threshold of increased internal (Burdon et al., 1982) or external (Burki et al., 1978; Gottfreid et al., 1981; Wiley and Zechman, 1966) resistance depends on the change in the background resistance rather than the absolute value of the added load. Thus, the background resistance plays an important role in the perception of respiratory mechanical loads. These mechanical changes may be mediated by afferents from the mouth, airways and respiratory muscles. The detection threshold has been determined as the load that is detected 50% of the time (Bennet et al., 1962; Campbell et al., 1961; Wiley and Zechman, 1966). Different threshold values have been shown to be most reliable when they are expressed as a fraction of the background. The detection threshold has been shown to be constant in normal subjects with externally elevated background (Stubbing et al., 1981) and asthmatic patients with internally increased mechanical loads (Burki et al., 1978). Breathing against mechanical loads induces changes in respiratory mechanics and there are pathways to mediate afferent information from respiratory mechanoreceptors to the cortex. However, the effect of the background resistance to the cortical activation of a loaded breath is unknown. Poor perception of respiratory effort in patients with chronic pulmonary disease may be due to a deficit in afferent information processing originating from respiratory

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17 mechanoreceptors or the pathways to the somatosensory cortex. It has been reported that the perceived magnitude declines with a background resistive load of 14.2 cmH2O (Burdon et al., 1983). Decreased perception of inspiratory resistive loads occurred when a higher background load were present to the subjects (McCloskey, 1973). It is likely that load detection may only require the activation of the sensory pathway but the magnitude estimation may involve evaluation of information from multiple afferent pathways. Evidence for the somatosensory cortex involvement in the mediation of signal detection has been investigated with the use of the EEG (Hillyard et al., 1971; Wilkinson and Seales, 1978). They have demonstrated the relationship between evoked potentials and measures of signal detection in different sensory modalities. Measurement of the RREP provides a unique way to investigate cortical activity involved in the perceptual responses to external mechanical loads. Asthma and other chronic obstructive pulmonary diseases are often associated with an increased airway resistance (like adding a background resistive load) and are known to decrease the perception of added resistive loads. Perception of inspiratory loads was blunted by the increased background airway resistance (Revelette et al., 1984). The short-latency component of evoked potentials reflects the integrity of the primary sensory pathways and varies in morphology with stimulus parameters such as intensity and frequency. The long-latency components of evoked potential reflect cognitive and psychological state of the subject. They are influenced primarily by factors extrinsic to the physical stimulus, such as arousal, attention, motivation, and task difficulty. Although it has been reported that the RREP can be elicited by different magnitudes of resistive loads, no study has addressed the relationship between the effect of increased background

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18 resistance on the conscious sensation of resistive inspiratory loads and the RREP. It is unknown if the decreased perception of added loads in the presence of an increased background resistance is associated with a change of RREP peaks. Summary of Experiments Cortical activation by respiratory stimuli has been recorded in both human and animals. Animal studies have demonstrated that afferents in the respiratory system have projection pathways to the cerebral cortex. Respiratory mechanical changes associated with loaded breaths are mediated via those afferent pathways. Measurement of the RREP provides a unique method to study cortical responses to respiratory load stimuli. The afferent pathway, cortical distribution and neural mechanisms processing respiratory mechanoreceptors have been only implied in human studies. However, perceptual process related to RREP responses and the relationship between the RREP elicited by respiratory mechanical loads and the SEP from other mechanical stimuli remains unknown. The RREP and SEP elicited by different somatosensory modalities (negative pressure, air puff, cutaneous stimulation) were tested in the first two parts of this dissertation. The RREP response to resistive loads with increased background resistance was investigated in the last part of this dissertation. If the somatosensory system is processing respiratory perceptual information, then comparing the latencies and amplitudes of evoked potentials elicited by respiratory load stimuli with known somatosensory stimuli will determine if respiratory mechanosensation is processed, in part, through the somatosensory cortex. The first two experiments were designed to determine the cortical localization and neural mechanism of the short-latency components (with peak latency < 100ms) of the RREP in healthy adults eliciting inspiration against mechanical loads. The short-latency peaks of the RREP were hypothesized as indicating the initial activation of the

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19 somatosensory cortex by breathing against the respiratory resistive load and were similar to mechanically elicited sensory evoked potentials (Hamalaimen et al., 1990). Rationale for Study 1 The activation of cortical neurons by respiratory mechanical loads has been studied using evoked potential techniques similar to those used in other somatosensory systems such as mechanical stimulation of the skin. The fact that subjects can sense the respiratory change against loads suggests that there must be activation of neurons in the cerebral cortex and this activation should be measurable. Therefore, the RREP provides unique neurophysiological evidence of cortical activation during mechanically loaded breathing. Revelette and Davenport (1990) found that the RREP was present bilaterally and can be elicited by occlusions either at the onset of inspiration or at mid-inspiration. They reported that the peak amplitude was greater and the peak latency shorter for the evoked potentials produced by the mid-inspiratory occlusions. The relationship of the RREP and mechanical load perception has been studied using graded resistive loads. Positive correlations were found between the peak amplitude of P1 and the magnitude of the added resistive load, and the magnitude estimation of the load (Knafelc and Davenport, 1997; 1999). A negative peak (Nf) occurred 25~40 msec after the loaded breath and was maximal at frontal electrode sites (Davenport et al., 1996). These topographical data provide evidence for dipole sources both pre (motor cortex) and post centrally (somatosensory cortex). It is difficult to determine the specific afferent mechanism mediating the perception of inspiratory mechanical loads because load-dependent changes are transmitted throughout most of the respiratory tract and pump. It has been hypothesized that the somatosensory system is mediating respiratory perceptual information processing.

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20 Comparing the neural markers for respiratory mechanical stimuli with known somatosensory stimuli (skin indentation) tests if respiratory mechanosensation is mediated, in part, by the same areas of the somatosensory cortex as mechanical stimulation of the skin. The results of this study will provide new information on the similarity between mechanical load neural activity related to the component peaks of the RREP and somatosensory mechanical skin stimulation. Although one report described the relationship between afferent activity in peripheral nerves and perception of the evoking mechanical stimulation of receptors in hand of human subject (Knibestol and Vallbo, 1979), it was unknown if or how the components of the SEP reflect the same processes as the RREP. Therefore, the present study investigated short-latency components in both the RREP and the SEP to identify the components showing a functionally similar distribution. The hypothesis tested in this part of the research: The RREP response to inspiratory occlusion was similar to the SEP elicited by cutaneous mechanical stimulation on hand. Rationale for Study 2 A cortical SEP can be recorded following air-puff stimulation of the hand (Schieppati and Ducati., 1984; Hashimoto, 1987; Hashimoto et al., 1990), face (Hashimoto et al., 1991) and foot (Hashimoto et al., 1992). These mechanical stimuli physiologically activate mechanoreceptors and are specific to mechanoreceptors unlike electrical stimulation usually employed to elicit SEPs (Gardner and Costanzo, 1980ab). Air-puff provides reproducible tap-like stimuli without steady pressure or direct contact to the skin. The air-puff stimulates a restricted area of skin in a spatial and temporal pattern and is known to activate cutaneous mechanoreceptors (Hashimoto et al., 1991b). Mouth mechanoreceptors have been proposed as one population of mechanoreceptors

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21 mediating the RREP. If this is correct, then the air puff mouth stimulation will elicit an evoked potential similar to the RREP. The cortical response to air-puff stimuli has been reported in monkeys (Gardener et al, 1984) and humans (Gardner and Tast, 1981). Therefore, air puff seems to provide an excellent method for eliciting neurophysiological activity underlying the evoked potentials (Hashimoto et al., 1991b; Hashimoto et al., 1992; Hashimoto et al., 1987). The SEP elicited by mechanical stimulation exhibited a gradual increase in amplitude and a decrease in latency with increasing stimulus intensity (Hashimoto et al., 1992). The SEP has been recorded in response to a variety of mechanical stimuli over the finger, palm, and face. The SEP elicited by mechanical stimuli were similar to those elicited by inspiratory occlusion. However, RREP studies with inspiratory occlusion have invariably documented latency delays and amplitude augmentation in early peaks relative to cutaneous mechanical counterparts. The latency delays and amplitude augmentation were ascribed to the global effect of mechanoreceptor activations and might be different sensory pathways processing the information. The second study investigated the mechanics of latency delay, amplitude augmentation and patterns of peaks for inspiratory occlusion elicited RREP by comparing this potential to evoked SEP with different sensory modalities from two selected areas (chest and mouth). The chest and mouth are chosen because they have mechanoreceptors that have a similar neural path length as respiratory muscles (chest) and they may be activated by inspiratory occlusions to elicit the RREP (mouth). If the pattern of the RREP is similar to that of SEPs with activation of the chest skin and mouth, then the latency

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22 and amplitude between the RREP and SEP should be similar. The hypothesis tested in this part of the research: The RREP response to inspiratory occlusion is similar to the SEP elicited by activation of mechanoreceptors on the chest wall (the level of diaphragm location, 7 th ribcage) and the buccal surface of the mouth. Rationale for Study 3 Perception of respiratory loads is dependent of the background conditions of the subjects respiratory tract and is blunted by elevated background airway resistance (Wiley and Zechman, 1966). Asthma and chronic obstructive respiratory disease is often associated with an elevated background airway resistance and is known to decrease the perception of added resistive loads. It is unknown if RREP peaks will decrease their amplitude or will not be elicited in the presence of an increased background resistance. Therefore, the third experiment investigated the short-latency RREP components elicited by inspiratory resistive loads with and without increased background load. The aim of the third study was to determine the RREP responses to inspiratory resistive load when gating by the elevated background resistance, and the relationship between conscious perceptual process of respiratory mechanical loads and RREP components. Short-latency components of the RREP were measured in subjects under two conditions: with and without increased background resistance. Two hypotheses were tested in this part of the research: Short-latency RREP components (P1, N1, and Nf) are involved in conscious perception of respiratory mechanical loads and are recorded only when the added resistive load is above the detection threshold, R 50 /R 0 is greater than 0.3.

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23 A resistive load detectable without increased background elicits an RREP but the RREP is not present when the background is increased to make the same load undetectable, R 50 /R 0 is less than 0.3. Summary of Hypotheses The hypotheses are summarized below: 1. RREP response to inspiratory occlusion is similar to the SEP elicited by cutaneous mechanical stimulation on hand. 2. RREP response to inspiratory occlusion is similar to the SEP elicited by activation of mechanoreceptors on the chest wall (the level of diaphragm location, 7 th ribcage) and the buccal surface of the mouth. 3. Short-latency RREP components (P1, N1, and Nf) are involved in conscious perception of respiratory mechanical loads and are recorded only when the added resistive load is above the detection threshold, R 50 /R 0 is greater than 0.3. 4. A resistive load detectable without increased background elicits an RREP but the RREP is not present when the background is increased to make the same load undetectable, R 50 /R 0 is less than 0.3.

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CHAPTER 2 THE CORTICAL RESPONSES TO INSPIRATORY LOADS AND TACTILE STIMULATION FOR HEALTHY ADULTS. Introduction The ability of the respiratory system to maintain normal ventilation when opposed by an external mechanical load is a motor process performed by the respiratory muscles acting as a pump to generate the driving force for air to flow and increase the lung volume. Application of extrinsic mechanical loads of sufficient magnitude alters this mechanical process and leads an activation of cortical neuron activities. The development of reliable objective neuronal methods for studying the sensory and motor mechanisms involved in respiratory load perception has resulted in a better understanding of the mechanisms of respiratory perception. Cortical activation by afferent stimulation is evidenced with event-related evoked potentials (ERP). The ERP elicited by visual, auditory and somatosensory stimuli have been well studied (Desmedt et al., 1987; Kekoni et al., 1992; 1997). The fact that subjects can sense a respiratory change against inspiratory loads suggests there must be activation of neurons in the cerebral cortex. The activation of cortical neurons by respiratory resistive loads has been studied using evoked potential techniques (Davenport et al., 1986). Davenport et al. (1986) applied inspiratory occlusions in healthy subjects and recorded the evoked potentials from the scalp. This study demonstrated that brief occlusion of the inspiratory airway in humans produces a series of respiratory related evoked potential (RREP) components. The RREP recorded over the somatosensory cortex provided a unique way to investigate higher cortical activities 24

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25 associated with inspiration against mechanical loads. Revelette and Davenport (1990) also found that the RREP was present bilaterally and elicited by occlusions either at the onset of inspiration or at mid-inspiration. They reported that the peak amplitude was greater and the peak latency shorter for the evoked potentials produced by the midinspiratory occlusions. Thus, the RREP provides a unique measure of respiratory load neural processing. A consistent pattern of three peaks of the RREP (Nf, P1, N1) has been identified (Davenport et al., 1986; Revelette and Davenport, 1990; Davenport et al., 1996; Knafelc and Davenport, 1997). The first peak of the RREP, P1, was a positive voltage that is elicited by the arrival of load activity from a population of afferents activated by the inspiratory occlusion stimulus depolarizing a column of neurons in the somatosensory cortex. Both P1 and N1 peaks were found in the central and post-central electrodes which correlated with the somatosensory region of the cortex (Davenport et al., 1996). The amplitudes and location of P1 and N1 peak activity suggest that the somatosensory cortex is one part of the neural pathway sub-serving respiratory load information processing (Logie et al., 1998). Neither the P1 nor the N1 peak was recorded from frontal electrodes. A negative peak, Nf, was recorded from the pre-central electrodes 25-40 ms after the occlusion (Davenport et al., 1996). This short latency frontal peak of the RREP may be parallel to the processing of P1 peak information. Human skin contains receptors that inform the brain about mechanical stimuli such as touch, pressure, and vibration (Mountcastle, 1980). The activation of somatosensory neuronal activity has been studied by recording the sensory evoked potentials (SEP) associated with mechanical or electrical stimuli (Onofrj et al., 1995; Desmedt et al., 1983;

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26 1984; Deiber et a., 1986; Gardner et al., 1984; Gerber and Meinck, 2000). Onofrj et al. (1995) reported that short-latency SEP components recorded following mechanical stimuli were of longer latency and smaller amplitude than evoked potentials recorded following electrical stimuli. This result was supported by the findings from Buchthal (1980) that more time was needed for skin indentation and processes of receptor transduction with mechanical stimuli than electrical stimuli. Studies of the SEP have focused on the issue of sources and components of SEP that reflect both cortical and subcortical activation of the brain (Larrson and Prevec, 1970; Desmedt et al., 1983, 1984; Kekoni et al., 1997). The SEP is the net result of activity in the somatosensory cortex in response to the afferent inputs. The SEP is correlated with physiological measures and used as method for measuring both electrophysiological and the perceptual events (Franzn and Offenloch, 1969; Gescheider and Wright, 1968; Hashimoto et al., 1988; 1992). The early components of the SEP following mechanical stimulation were reported to correlate with stimulus magnitude and magnitude estimation (Hashimoto et al., 1987; 1992). The activation of cortical neurons by respiratory mechanical loads has been studied using evoked potential techniques similar to those used in recording the SEP. The afferents mediating load perception are primarily located in the respiratory muscle pump (Davenport et al., 1993). However, it is difficult to determine the specific afferent mechanism mediating the respiratory afferent information because load-dependent changes are transmitted throughout most of the respiratory tract and pump. The scalp was mapped for the distribution of the short latency peaks of the RREP using the earlobe reference. The P1 peak was found predominately over the somatosensory

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27 region of the cortex in both animals (Davenport et al., 1985; Davenport and Hutchison, 2001) and humans (Davenport et al., 1986; Logie et al., 2000). The RREP associated with inspiratory occlusion was recorded bilaterally over the somatosensory cortex and was also elicited by occlusions either at the onset of inspiration or at mid-inspiration (Davenport et al., 1986; Revelette & Davenport, 1990). The P1 peak amplitude was greater and the peak latency shorter for the evoked potentials produced by the midinspiratory occlusions. These reports provide evidence for dipole sources both pre (motor cortex) and post centrally (somatosensory cortex). Thus, respiratory afferents project via somatosensory pathways to the cerebral cortex. This is similar to somatosensory cortical processing of cutaneous mechanical information. Respiratory muscle afferents have transduction properties and cortical projection pathways consistent with their hypothesized role in respiratory load sensation. Logie et al. (1998) identified the dipole locations of the P1 peak bilaterally located within the primary somatosensory and the Nf peak bilaterally in the supplementary motor cortex. Activation of the contralateral sensorimotor cortex was reported following stimulation of the intercostal muscle afferents (Davenport et al., 1993; Gandevia and Macefield, 1989) and stimulation of phrenic afferents (Davenport et al., 1985). If this respiratory related cortical activity is similar to evoked potentials for other sensory system, then the early P1 potential represents the arrival of the sensory signal in the somatosensory region of the cerebral cortex. The study of the RREP has focused on the short-latency components (P1, Nf and N1) because they were similar to the initial sensory components in the cortical processing of respiratory resistive load information (Davenport et al., 1986). The main function of

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28 the somatosensory cortex is to integrate mechanical information from the skin, joint, and muscles afferents. Both RREP and SEP elicited by mechanical stimuli represent cortical neural activity with different scalp distributions. However, the correlation between RREP and the mechanical induced SEP is unknown. The purpose of the present study was to compare the inspiratory occlusion elicited RREP and the SEP elicited by cutaneous mechanical stimulation. The SEP and RREP were recorded in the same individuals. It was hypothesized that the P1 peak of the RREP and SEP are recorded over the somatosensory cortex. It was further hypothesized that the latency of the SEP elicited by mechanical stimulation is shorter than the RREP elicited by inspiratory occlusions. Methods Subjects Adult subjects (9 male, 11 female; average age = 25.5) were recruited. The subjects were healthy and normal on the basis of no self-reported history of cardiorespiratory disease, no self-reported history of smoking and no evidence of current major or minor illness. They were informed of the nature of the study before starting the experiment and written consent was obtained. The institutional Review Board of the University of Florida reviewed and approved this study. Pulmonary Function Testing The subjects were asked to refrain from strenuous physical activity, large meals and caffeine for at least four hours prior to the test. Subjects were seated upright in a chair. Spirometry testing conformed to the American Thoracic Society Standards. Forced vital capacity (FVC), forced expiratory volume within 1 second (FEV1) and airway resistance by the forced oscillation method were measured in each subject. A FEV1 greater than 70 % predicted was required for the subjects to continue in the study.

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29 RREP The subject was seated in a sound insulated room, with the back, neck and head comfortably supported. An electrode cap with integral electrodes was used to record scalp EEG activity. The cap was placed on the subjects head, positioned and secured with a strap. Scalp and electrode contact was made by the application of electro-conducing paste administered through the center opening in the electrode. The impedance levels for each electrode was checked and maintained below 5 K. The electrode cap was then connected to an electroencephalograph system (Model 12 Neurodata Acquisition System, Grass Instruments, Quincy, MA). The EEG activity was recorded from electrode positions; Fz, F4, F3, Cz, C4, C3, Cz', C4', C3', Pz, P4, and P3 according to the International 10/20 system with the joined earlobes as the reference. EEG activity was amplified and bandpass filtered (0.3Hz-1 kHz). Eye movements (EOG) were recorded with electrodes placed over the lateral edge of the eye. The EEG activities and mouth pressure (Pm) were recorded and led to a signal analysis system (Signal 2, Cambridge Electronics Ltd). The experimenter and recording equipment were in the adjacent room. The subject was monitored with a video camera during the experiment. The subject respired through a non-rebreathing valve with the inspiratory port connected to a pneumotachograph and occlusion value. Mouth pressure was recorded at the center of the non-rebreathing valve, sensed with a differential pressure transducer and amplified with a signal conditioner. The inspiratory load was presented by silently inflating the occlusion valve at approximately the mid-inspiration point of the breath. Pm was used for timing of respiratory occlusion. There were 2 experimental trials for the RREP: control trial and load trial. Both trials were separated by a 5-minute rest period off the breathing apparatus. During the load trial, inspiration was occluded for about 300

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30 msec every 2-5 breaths. A minimum of 100 occlusions were presented. The control trial consisted of 100 valve activations every 2-5 breaths. The subjects inspired through a side port on the occlusion manifold and the inspiration was not obstructed with the valve activations. The subject watched a video movie to mask the auditory cues. The RREP was obtained by averaging a minimum of 64 occlusions. An individual occlusion presentation was included in the average if it produced a minimum 3 cmH2O change in Pm and there was no eye blink. The early RREP components were defined as follows: P1 was the maximal positive deflection 40-60 milliseconds following the onset of the change in Pm. N1 was the negative deflection in the latency range of 80-120 milliseconds; Nf was the negative deflection in the latency range of 25-40 milliseconds. Baseline-to-peak amplitudes were determined for each peak. Latencies were determined as the time from the onset of the Pm change to the RREP peak. The control trial used the same procedures except there was no occluded breath. SEP Cutaneous mechanical stimulation consisted of a square wave indentation of the skin on the subjects non-dominant hand. The skin indentation was produced by a controlled linear induction motor attached to a blunt flexible tube placed 2mm above the surface of the skin. To obtain the SEP, subjects were asked to place their forearm on a handrest in a fixed position. The hand was placed in position with the hand and mechanical stimulator screened from the subject by a curtain. A series of 512 cutaneous stimulations were delivered. A strain gauge was used to calibrate mechanical pulses. Each skin indentation had a duration of about 400 msec and was delivered every 1 second. The subject watched a videotape movie. There were 2 cutaneous stimulation trials. The first trial was mechanical stimulation of the skin. The second trial was

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31 activation of the stimulator but the skin was not indented. The order of the trials was counterbalanced across subjects. The SEP was analyzed in the same manner as the RREP. EEG activity was recorded from scalp electrodes referenced to joined-earlobes. EOG was recorded and any presentation containing an eye-blink was rejected. A minimum of 256 cutaneous stimuli were averaged for each subject. Two SEP components elicited by cutaneous stimulation were defined as follow: P1 was the maximal positive deflection 20-30 milliseconds following the onset of skin indentation and N1was the negative deflection 50-70 milliseconds after the onset of skin indentation. Baseline-to-peak amplitudes were determined for each peak of the SEP. Latencies were obtained as the time from the onset of the indentation to the SEP peak. A total of 2 RREP and 2 SEP trials were presented in this study. Statistical Analysis One-way repeated measures analysis of variance (ANOVA) was used to determine differences between SEP and RREP components. When the ANOVA showed a significant group difference, a multiple comparison procedures was used to test for the differences between latencies and amplitudes for each RREP and SEP peak. The statistical significance was determined at p < 0.05 level in this study. Result The RREP elicited by inspiratory occlusion was observed bilaterally and was absent with unoccluded breaths. The P1 peak of the RREP was identified at C4' and C3' scalp sites (Fig. 2-3). The N1 peak of the RREP was found at C4' and C3' scalp sites (Fig. 2-3). Cutaneous stimulation applied to middle finger of the subjects non-dominant hand

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32 elicited the P1 peak at C4' scalp site (Fig. 2-2) in all subjects. The SEP N1 peak was obtained in the C3' and C4' scalp sites (Fig. 2-2). The P1 and N1 peaks were observed bilaterally in the RREP (Fig. 2-3) but unilaterally in SEP (Fig 2-2). There was no significant difference in the RREP P1 peak latency and amplitude between right (C4') and left (C3') cortices. There was no significant difference in the RREP N1 peak latency and amplitude between right (C4') and left (C3') cortices. The SEP P1 peak amplitude was significantly greater in the C4' electrode site than the C3' electrode site (Fig. 2-6). The SEP N1 peak amplitude was significantly greater in the C4' electrode site than the C3' electrode site (Fig. 2-6). The averaged latency and amplitude for the RREP and SEP peaks are presented in Table 2-1. The RREP peak latencies of the P1 and N1 were 63.22 + 0.3 ms and 107.53 + 0.3 ms, respectively. The SEP peak latencies of the P1 and N1 peaks were 27.41 + 1.3 ms and 42.69 + 0.9 ms, respectively. The peak latencies of the P1 and N1 RREP peaks were significantly longer than the corresponding peaks of the SEP (p < 0.05). The peak amplitudes of the P1 and N1 RREP peaks were 2.18 + 1.49V and -3.80 + 2.11V, respectively. The SEP peak amplitudes of P1 and N1 were 0.56 + 0.30 V and -0.48 + 0.23 V, respectively. The P1 and N1 peak amplitudes of the RREP were significantly greater than the corresponding peaks of the SEP (p < 0.05) (Fig. 2-5). The Nf peak was observed in the RREP but not observed in the SEP (Fig 2-4). The peak amplitude of the Nf peak was -2.51 + 1.24V. The peak latency of the Nf peak was 25.26 + 5.1ms. Discussion The similarity of the RREP waveform to that observed in response to mechanically elicited SEP was demonstrated in the present study. The P1 peak of the RREP peaks was observed bilaterally, whereas, the analogous peak of the SEP was observed contralateral

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33 to the stimulated hand. The scalp distributions of the RREP peaks were similar to the SEP at the electrode positions that are over the somatosensory cortex. The latencies of the P1 and N1 peaks of the RREP were significantly longer than those of the SEP (p < 0.05). The amplitudes of the P1 and N1 peaks of the RREP were significantly greater than those of the SEP. The Nf peak was only observed in the frontal scalp sites of the RREP. Inspiratory occlusion is a mechanical load that alters the afferent activity of several populations of respiratory mechanoreceptors in the mouth, pharynx, larynx, lung and respiratory muscles. The inspiratory occlusion halts the normal changes in inspiratory airflow and lung volume. This change in breathing pattern results in a change in the afferent activities throughout the respiratory system. The RREP waveform elicited by inspiratory occlusion was similar to the SEP elicited by mechanically stimulating cutaneous afferents. The SEP from the present study is consistent with previous studies (Kakigi and Shibasaki, 1984; Mima et al., 1996). The latency for mechanical stimulation SEP is slower than electrical stimulation due to the time required for skin indentation and receptor transudation. In the present study, the RREP peaks elicited by inspiration against an occluded airway had significantly longer latencies and larger amplitudes than the SEP (p < 0.05). The difference between the RREP and SEP latencies may due to a longer transduction time required for respiratory mechanoreceptors to activate the respiratory afferents. The difference between the RREP and SEP amplitudes may be due to more respiratory afferents activated and respiratory mechanical change activating afferents from both sides of the respiratory tract. The dorsal column-lemniscal system mediates the afferent pathway to the somatosensory cortex for the SEP (Williams, 1981). The SEP is elicited by activating

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34 afferent fibers that travel through multiple relays via the thalamus to the somatosensory cortex (Horne and Tracey, 1979). Two ascending system may be involved to the relay of sensory information to the somatosensory cortex: (1) the lemniscal pathway and (2) the extra-lemniscal reticular pathway (Albe-Fessard and Bowsher, 1965). The lemniscal pathway includes the ventroposterolateral (VPL) nuclei and ventral basal nuclei in the thalamus. The extra-lemniscal pathway includes the brainstem reticular formation and ventral basal nuclei in the thalamus. Jasper (1949) reported that short-latency evoked potentials are produced by impulses traveling via the lemniscal pathway whereas; long-latency evoked potentials are produced by impulses traveling via extra-lemniscal pathway. The latency for the SEP in the present study is consistent with the lemniscal pathway. The activation of a somatosensory modality projects to the contralateral somatosensory cortex (Kekoni et al., 1992; McLaughlin and Kelly, 1993; Nagamine et al., 1998). Recordings of cortical responses to afferent stimuli confirm that the electrical responses at 50 ms and 100 ms latencies are generated by activity in the SI and SII somatosensory cortices (Hari, 1980). The SEP evoked by electrical stimulation of median nerve at the wrist or the index finger has a similar cortical distribution (Onofrj et al., 1990; Grisolia and Wiederholt, 1980). The SEP has been recorded with electrical stimulation of the phrenic nerve (Zifko et al., 1995). The amplitudes of the phrenic SEP were greatest in the postcentral gyrus (somatosensory region), which lies just posterior to the diaphragmatic cortical representation reported by Colebatch et al. (1991). This is also supported by the recordings from the exposed cortex in human demonstrating that the short latency peaks of the SEP were generated in the primary somatosensory cortex

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35 (Allison et al., 1980). The generation of short-latency SEP components reported by Deiber et al. (1986) and Desmedt et al. (1987) demonstrated that area 3b of the somatosensory cortex was activated by afferents from VPL via thalamocortical pathway. Several variables have been found to alter the hand mechanical SEP. Variation in the rise time of the stimulus alters the rate of mechanical indentation of the skin and thus the transduction process (Johansson et al., 1979b; Jarvilehto et al., 1981). The amplitude is dependent on the stiffness of the indentation probe, the surface area stimulated, the depth of the indentation and the indentation duration. In the present study, a square wave rapid onset indentation was used for focal stimulation with mild indentation depths to elicit a reproducible cutaneous SEP. The P1 peak of the RREP was observed to be of greatest amplitude in the C3'and C4' electrode sites. These sites are caudal to the central sulcus (C3 and C4) and are over the post-central gyrus, the primary somatosensory region. This is consistent with the P1 peak of the SEP elicited by mechanical stimulation of the digits (Pratt and Starr, 1981). The P1 peak of the SEP in the present study had a latency of 20-25 milliseconds. The recording electrode sites were over the post-central gyrus lateral to the midline which is consistent with the somatotopical representation of the fingers in the primary somatosensory cortex. Thus, the mechanical SEP and the RREP P1 peaks are consistent with an activation of neurons in the primary somatosensory region of the cerebral cortex. The N1 peak of the RREP was observed to be of greatest amplitude in the central electrode sites. These electrode sites were over the post-central gyrus of the cerebral cortex which is consistent with the somatotopical representation of the fingers in the sensorimotor cortex (Pratt and Starr, 1981). In addition, Webster and Colrain (2000)

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36 reported that the N1 peak is recorded from the vertical dipole source and is affected by the attention. Thus, the N1 peak of RREP may be indicative of second order cortical processing of respiratory afferent information. The N1 peak of the SEP with a latency of 40-50 milliseconds was elicited by the cutaneous stimulation. The electrode sites were also over the post-central gyrus of the cerebral cortex (Pratt and Starr, 1981). Thus, the mechanical SEP and the RREP N1 peaks are consistent with an activation of neurons in the sensorimotor region of the cerebral cortex. The Nf peak was observed 25-40 ms after the onset of inspiratory occlusion and was only found in the frontal electrode sites of the RREP. These electrode sites were over the pre-central gyrus, the prefrontal region of the cerebral cortex. Thus, the Nf peak is unique to the RREP and may be related to the feed-forward preparation of the respiratory behavioral motor response. In the current study, the P1 and N1 peaks of the RREP and SEP were observed in the same subject. The difference between the RREP P1 and N1 amplitudes and the SEP amplitudes suggests that the magnitude of neural activity is correlated with the magnitude of stimulus. The smaller amplitude of the evoked potential recorded with cutaneous mechanical stimulation compared to the RREP suggest that inspiratory occlusion activates more afferents than focal mechanical stimulation of the skin. Conclusion Multi-channel evoked potential recordings have provided a measurement of cortical neural activity associated activation of respiratory and cutaneous mechanoreceptors. Mechanically stimulated cutaneous somatosensory evoked potentials has a maximal response over the cortex contralateral to the side of stimulation. The scalp distribution of RREP was bilateral but similar to the cortical regions of mechanically elicited SEP.

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37 RREP P1 and N1peaks were found to correspond to those of the SEP. The results of the present study suggest that the P1 peak of the RREP and the analogous P1 peak of the SEP activated neurons in the somatosensory cortex. Thus, the P1 and N1 peaks of the RREP are analogous to the SEP P30 and N50 peaks. The RREP, however, has a unique activation of the pre-frontal cortex, Nf peak, which is not found in the SEP. The exact sensory pathways mediating somatosensory processing of the respiratory afferent information remain unknown. The differences in amplitude suggest a greater population of respiratory afferents is activated by inspiratory occlusion than focal indentation of the skin. The longer latencies of the RREP peaks suggest a longer time is required for changes in airway mechanics to stimulate respiratory mechanoreceptors.

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38 Table 2-1 Mean amplitude and latency data for the respiratory-related evoked potential and the somatosensory evoked potential P1 average at C3' P1 average at C4' N1 average at C3' N1 average at C4' AmplitudeV RREP 2.08 1.53 2.18 1.49 -3.68 2.02 -3.80 2.11 SEP 0.30 0.16 0.56 0.30 # -0.33 0.17 -0.48 0.23 # Latency msec RREP 63.22 0.3 107.53 0.3 SEP 27.41 .3 42.69 0.9 indicates a significantly different between the RREP and SEP. # indicates a significantly different between C3' and C4'.

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39 monitor Digital video camera Movie Pressure SOUND ISOLATED ROOM Signal Acquisition and Data Analysis occlusion RREP Figure 2-1. Respiratory-related evoked potential Schmidt

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40 C3' 5 V N1 P1 C4' Stimulus 0.1 0.3 0.4 0.2 0 sec Figure 2-2. SEP at C4' and C3' electrode sites (C4'Right hemisphere; C3'-Left hemisphere).

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41 C3 N1 P1 5 V C4' Stimulus 0.2 0.6 0.8 0 0.4 sec Figure 2-3. RREP at C4' and C3' electrode sites (C4'-Right hemisphere; C3'-Left hemisphere).

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42 0.3 sec F4 R R E P RREP N Nf f 5V F4 SEP Stimulus 0 0.1 0.2 Figure 2-4. The RREP and SEP at F4 electrode site. The Nf peak was only observed in the RREP but not in the SEP.

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43 Amplitude (V) -8-6-4-20246 RREPP1SEPP1RREPN1SEPN1 # # Figure 2-5. Comparison for the amplitudes of the P1 and N1 peaks of the RREP and the SEP. The amplitude of the P1 peak of the RREP was significantly greater than the SEP (p < 0.05). The amplitude of the N1 peak of the RREP was significantly greater than the SEP (p < 0.05). # indicates a significantly different between the RREP and the SEP.

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44 Amplitude (V) -8-6-4-20246 RREP P1C4'C3'C4'C3'C4'C3'C4'C3'RREP N1SEP P1SEP N1** Figure 2-6. Comparison between the C3' and C4' electrode sites for the P1 and N1 peaks of the RREP and the SEP. The SEP P1 peak was significantly greater amplitude in the C4' electrode site (p < 0.05). The SEP N1 peak amplitude was significantly greater in the C4 electrode site (p < 0.05). There were no significant differences in the RREP P1 and N1 peak amplitudes between the C4' and C3' electrode sites.* indicates a significantly different between the C3' and C4' electrode sites.

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CHAPTER 3 CORTICAL EVOKED POTENTIALS ELICITED BY INSPIRATORY OCCLUSION, AIR-PUFF, AND CUTANEOUS STIMULATION IN HEALTHY ADULTS Introduction Activation of mechanoreceptors by mid-inspiratory occlusion elicits the RREP (Revelette and Davenport, 1990). Activation of the intercostal muscles (Gandevia and Macefield, 1989), vagal afferents (Tougas et al., 1993), phrenic afferents (Straus et al., 1997; Zifko et al., 1995), and laryngeal afferents of the upper airway (Yin et al., 1997; Daubenspeck et al., 2000) are known to elicit cortical evoked potentials. These respiratory afferent sources may contribute to the RREP. The early peaks of RREP are an indicator of respiratory sensory information arriving to the cortex (Davenport et al., 1986; Logie et al., 1998; Colrain et al., 2000). The previous study indicated a similarity in cortical activation between the RREP and the SEP (Davenport et al., 1996; Logie et al., 1998). There were differences in the latency, amplitude and bilateral cortical activation. It remains unknown if these differences are due to the sources that generate the RREP, the neural path length and/or the cortical distribution of sensory activity. In normal adult mammalian sensory systems information from specific sensory modalities is transmitted along parallel, modality-specific, multisynaptic pathways (Mountcastle, 1980). The cortical projection of human mechanoreceptors has been identified for skin mechanoreceptors. Information transduced by sensory receptors is relayed via the spinal cord to thalamus, then project to specific areas of the cortex. The primary somatosensory cortex has a somatotopic modality-specific organization (Kaas, 45

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46 1991). Area 3b and 1 of the somatosensory cortex are mainly activated by cutaneous stimulation, while area 3a receives afferent input from deep sensory receptors and area 2 plays a somatosensory integrative function. The SEP elicited by mechanical stimuli was similar to those induced by inspiratory occlusion. The SEP elicited by mechanical stimulation exhibited an increased of amplitude and decreased of latency with increasing stimulus intensity (Hashimoto et al., 1992). RREP studies with inspiratory occlusion have reported longer latencies and greater amplitudes for the P1 and N1 peaks when compared with the SEP elicited by mechanical stimuli. The latency delays and amplitude difference may be due to the number of mechanoreceptor populations activated or may be due to different sensory pathway processing of respiratory information. Intercostals muscle afferents and the phrenic afferents project to the somatosensory cortex (Gandevia and Macefield, 1989; Davenport et al., 1993; Zifko et al., 1995). In addition, cortical evoked potentials have been elicited by vagal stimulation (Tongus et al., 1993), inspiratory occlusion (Davenport et al., 1986; Davenport et al., 2000), and expiratory occlusion (Hammond et al., 1999). These findings suggest that respiratory sensation is the result, in part, of the cortical integration of multiple respiratory afferents projecting to the somatosensory cortex. Mechanoreceptors of the respiratory tract that can be altered by an inspiratory occlusion are in the upper airways, the lung, the chest wall and the respiratory muscles. The present study was designed to investigate the mechanism of the latency, amplitude and patterns of RREP peaks. Inspiratory occlusion elicited RREP was compared to mechanically elicited SEP with different modalities of stimulation from two selected

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47 areas, chest wall and mouth. Thus, the purpose of this study was to compare the early peaks of the RREP with the SEP activated by mechanical stimuli in the respiratory related areas. If the pattern of the RREP is similar to the SEP with specific mechanical activation, then it was reasoned that those mechanoreceptors may mediate the RREP. However, if differences in the latencies and amplitudes occur between the RREP and the SEP, then the mechanoreceptors in these selective areas of stimulation are not the primary source of the RREP. Therefore, it was hypothesized that the latencies and amplitudes of short-latency RREP components are similar to those of the mouth SEP and chest SEP. Method Subjects Twenty normal subjects were recruited (11 male, 9 female; average age = 22.35 years) for this study. Subjects were classified as normal on the basis of self-reported habitual good health, no self-reported history of cardiorespiratory disease, history of smoking nor evidence of current major or minor illness. Forced vital capacity (FVC) and forced expiratory volume within 1 second (FEV1) and airway resistance were measured in each subject. The Institutional Review Board of the University of Florida reviewed and approved this study. The study was explained to the subject and written consent obtained. Pulmonary Function Testing Subjects were asked to refrain from large meals and caffeine for at least four hours prior to the experiment. Subjects were seated upright in a chair to perform the spirometry testing that conformed to American Thoracic Society Standards. FVC, FEV1 and airway resistance were collected by a computerized spirometer and impulse oscillometer system.

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48 RREP Elicited by Inspiratory Load Subjects were seated in a sound insulated room, with the back, neck and head comfortably supported. An electrode cap with integral electrodes was used to record scalp EEG activity. The electrode positions were based on the International 10-20 System. The cap was placed on the subjects head, positioned and secured with a strap. Scalp and electrode contact has been made by the application of electro-conducing paste administered through the center opening in the electrode. The impedance levels for each electrode were checked and maintained below 5 k. The electrode cap was connected to an electroencephalograph system and EEG activity recorded. Two electrodes were placed over the lateral edge of the eye for recording EOG. Any load presentation that occurs with an eye-blink was discarded. The EEG activity was bandpass filtered, amplified and led into an on-line signal averaging computer system (CED 1401, Cambridge Electronics Ltd., Cambridge, England). The EEG activity for each presentation was stored on computer disk. The experimenter and recording equipment were in the adjacent room. The subject was monitored with a video camera. The subject respired through a non-rebreathing valve with the inspiratory port connected to the loading manifold. Mouth pressure was sensed with a differential pressure transducer and signal conditioner. The inspiratory load was presented by silently inflating the occlusion valve at approximately the mid-inspiration point of the breath. Each loaded breath was separated by 2-6 unloaded breaths. A TTL pulse generated by the inspiratory occlusion valve controller triggered the collection of 50ms of pre-trigger and 950ms of post-trigger EEG and mouth pressure (Pm) data. The duration of each occlusion was about 200ms. There were two respiratory experimental trials for the RREP: control trial and load trial. Both trials were separated by a 5-minute rest period off the breathing

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49 apparatus. During the load trial, the subject was watching a pre-recorded videotape cartoon movie with the sound masking experimental noises. One hundred loaded breaths were presented in each trial. The control trial used the same protocol, except that there were no occluded breaths presented. The recorded data for each presentation were evaluated for inclusion in the averaged RREP. The baseline EEG traces were DC corrected if necessary. The inclusion and exclusion criteria were the same as the previous study. The presence, latency and amplitude of RREP peaks: Nf, P1 and N1 were determined for each scalp location from the averaged EEG traces. Peak latencies were measured as the time from the onset of the change in mouth pressure, to the EEG peak. The zero-to-peak amplitude was measured at the peak of each component. The RREP peaks were identified in the following manner: P1 was defined as the positive peak within a latency window of 40-70 ms, Nf was defined as the negative peak within a latency window of 25-40 msec, N1 was defined as the negative peak within a latency window of 80-120 msec. The site with the largest amplitude and shortest latency for each peak was determined. Mechanical Stimulation of the Chest and the Mouth Cutaneous mechanical stimulation was performed on the subjects chest wall between the 6 th and 7 th ribs. This site was chosen because the nerve path length to the cortex is approximately equal to the phrenic nerve and intercostals muscles. The skin indentation was produced by a controlled linear induction motor attached to a blunt flexible tube placed 2mm above the surface of the chest wall. The probe tube was in a plastic sleeve that was placed in contact with the skin of the chest wall and fixed in position with a harness. The mechanical stimulator was screened from the subject by a

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50 curtain. A series of 512 cutaneous stimulations were applied by linear displacement of the probe tube. Each indentation had a duration of 400 ms and was presented every second. The buccal surface of the cheek was mechanically stimulated with air pulses. Air puff stimulation has been used to elicit cutaneous SEP in humans. The air puff provides reproducible mechanical stimuli without direct contact to the skin and permits stimulation of a restricted area of the buccal surface. Air puff mechanical stimulation was delivered by a tube puffing air onto the cheek of the buccal surface of the mouth. Air puff stimulation was delivered through the tube with one end fixed in the mouthpiece, the tip extending 2-5 mm lateral to the molars. The other end of the tube was connected to the pressure source. The air puff had a rise time of 0.2ms and duration of 300 msec and was driven by oxygen gas at 20 cmH2O. The cross-sectional area covered by the air puff was about 3.6 mm 2 Each trial consisted of 256 stimuli delivered every second to the buccal surface of the mouth. During the mechanical stimulation sessions the subject watched a videotape cartoon movie and did not attend to the stimulus. There were 2-paired sessions (4 trials) for chest wall and mouth mechanical stimulation. The first chest trial consisted of cutaneous mechanical stimulation. The second chest trial consisted of activation mechanical stimulator but the skin has not been indented. The first mouth trial consisted of positive pressure pulses applied to the buccal surface of the cheek. The second mouth trial consisted of the activation of the positive pressure solenoid but no air was delivered. The order of the trials was counterbalanced across subjects. The SEP elicited by air puffs delivered to the mouth and the SEP elicited by mechanical stimulation of the chest was analyzed in the same manner as the RREP. EEG

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51 activity was recorded from scalp electrodes referenced to the joined earlobes. EOG was recorded and any presentation containing an eye-blink was rejected. The SEP and RREP peaks were identified. Both the latency and amplitude of each peak were determined for each stimulus condition (respiration, mouth or chest). Statistical Analysis The descriptive statistics of all variables in the study were calculated and expressed as mean standard deviation (SD). One way analysis of variance (ANOVA) for repeated measurement was conducted to compare the latencies and amplitudes of evoked potentials. The repeated measures ANOVA was performed for each peak with different modalities of evoked potentials. When the ANOVA showed significant differences in peaks, a multiple comparison (Bonforoni correction) was used to test for pairwise differences. The significant difference level of the study is set at p < 0.05. Result The P1, N1 and Nf peaks of the RREP were identified in all subjects (Fig 3-1. and Fig 3-2.). The P1 and N1 peaks were identified in the mouth SEP and chest SEP (Fig 3-3. and Fig 3-4.). The RREP elicited by inspiratory occlusion was observed bilaterally and was absent with unoccluded breaths. The P1 peak of the RREP was identified at C4' and C3' electrode sites (Fig. 3-2). The N1 peak of the RREP was found at C4' and C3' electrode sites (Fig. 3-2). The P1 peak of the mouth SEP was found at C4' electrode site (Fig 3-3). The N1 peak of the mouth SEP was found at C4' and C3' electrode sites (Fig 3-3). The P1 peak of the chest SEP was found at C4' electrode site (Fig 3-4). The N1 peak of the chest SEP was found at C4' and C3' electrode sites (Fig 3-4). The Nf peak was only found in the F4 electrode site of the RREP but not I the F4 electrode site of the mouth SEP and chest SEP (Fig 3-1).

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52 The latencies and amplitudes for the P1 and N1 peaks of the RREP, mouth SEP and chest SEP are presented in Table 3-1. The P1 peak latency of the RREP was 63.49 + 6.5 ms. The N1 peak latency of the RREP was 107.30 + 11.8 ms. The P1 peak latency of the mouth SEP was 39.91 + 3.7 ms and significantly shorter than the RREP P1 peak. The N1 peak latency of the mouth SEP was 76.10 + 6.1 ms and significantly shorter than the RREP N1 peak. The P1 peak latency of the chest SEP was 31.00 + 4.6 ms and significantly shorter than the RREP P1 peak but not significantly different form the mouth P1 peak. The N1 peak latency of the chest SEP was 54.17 + 2.8 ms and significantly shorter than the RREP P1 peak but not significantly different from the mouth P1 peak. The P1 peak amplitude of the RREP was 1.9 + 1.2 V. The N1 peak amplitude of the RREP was -4.15 + 2.6 V. The P1 peak amplitude of the mouth SEP was 1.53 + 0.1 V. The N1 peak amplitude of the mouth SEP was -2.02 + 1.4 V. The P1 peak amplitude of the chest SEP was 1.19 + 1.1 V. The N1 peak amplitude of the chest SEP was -1.58 + 2.4 V. The amplitudes of the P1 peak were significantly greater for the RREP than the chest SEP (p < 0.05). However, there was no significant difference for the P1 peak amplitude of the RREP and mouth SEP. The N1 peak amplitude of the RREP was significantly greater than the mouth SEP and the chest SEP (p< 0.05). There were no significant differences for the P1 and N1 peak amplitudes between the mouth SEP and chest SEP. Discussion The P1 and N1 peaks of the RREP elicited by inspiratory occlusion were analogous to those of the SEP elicited by either air-puff stimulation to the mouth or cutaneous stimulation to the chest. The P1 peak of the RREP was found bilaterally in the centroparietal scalp over the somatosensory cortex. The SEP P1 peaks were also found

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53 over the somatosensory cortex but were unilateral, contralateral to the stimulated side. The latencies of the P1 and N1 peaks of the RREP were significantly longer than those of the SEPs from either mouth stimulation or chest stimulation. The P1 and N1 peak amplitudes of the RREP were significantly greater than the corresponding peaks of the chest SEP. Only the N1 peak amplitude of the RREP was significantly greater than the N1 peak of the mouth SEP. Evoked potentials elicited by mechanical stimulation of the skin (Hari, 1980 Hashimoto et al., 1987) and respiratory system have been reported (Davenport et al., 1986). However, no study has compared the cortical evoked potentials from the skin and respiratory mechanical stimulation. The results from the present study showed that inspiratory occlusion activates cortical neurons in the somatosensory cortex which was also found for the SEP elicited by mouth air puff and cutaneous indentation. The RREP P1 peak had a bilateral localization while both the mouth SEP and chest SEP were lateralized. The P1 peak of RREP was observed to be of greatest amplitude in the C3'and C4' electrode sites. These electrode sites are over the post-central gyrus, the primary somatosensory region. This is consistent with the reports that the P1 peak of the RREP reflects the arrival of the afferent projection to the primary sensory area (Davenport et al., 1986; Davenport et al., 1996). The electrode sites of the P1 peak of the mouth SEP and chest SEP were over the post-central gyrus lateral to the midline which is consistent with the somatotopical representation of the fingers and mouth in the primary somatosensory cortex (Pratt and Starr, 1981). Thus, the mechanical SEP and the RREP P1 peaks are consistent with an activation of neurons in the primary somatosensory region of the cerebral cortex. Knafelc and Davenport (1997) reported that the P1 peak

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54 amplitude of the RREP was correlated to the magnitude of the added resistive load. This was similar to the relationship reported by Hashimoto et al. (1988) that the peak amplitude of the SEP elicited by air puff stimulation was correlated to the magnitude of the stimulus intensity. Thus, the mechanically elicited SEP and the RREP may have similar neural processing mechanism but different afferent origins. The N1 peak of RREP was observed to be of greatest amplitude in the Cz and Cz' electrode sites. These electrode sites were over the central sulcus at the midline of the cerebral cortex (Pratt and Starr, 1981). The electrode sites of the N1 peak of the mouth SEP and chest SEP were over the central sulcus at the midline of the cerebral cortex which is also consistent with the representation in the sensorimotor cortex (Pratt and Starr, 1981). Thus, the mechanical SEP and the RREP N1 peaks are consistent with an activation of neurons in the sensorimotor region of the cerebral cortex. The N1 peak of the RREP was the negative peak associated with the P1 peak of the RREP (Davenport et al., 1986). Bloch-Salisbury et al. (1998) reported that N1 peak amplitude of the RREP decreases as the resistive load decreased, similar to P1 peak of the RREP. The N1 peak amplitude also increases with attention to the load. Thus, the N1 peak of the RREP may be the second order cortical processing of the respiratory afferent information that is based on perceiving the first-order somatosensory information and may affect subsequent cognitive processing. The bilateral frontal Nf peak of the RREP was not present in chest or mouth SEPs. The Nf peak may reflect activity in prefrontal structures (Logie et al., 1998). Thalamocortical inputs to the somatosensory cortex project predominantly to deep cortical layers generate a surface positive initial potential (Wood and Allison, 1981;

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55 Wood et al., 1985). The frontal Nf peak was not due to the activity in the somatosensory cortex. It has been reported that the prefrontal cortex gated the early stage of somatosensory input and that it exerts inhibitory modulation on sensory processing (Yamaguchi and Knight, 1990). This may be the reason that the Nf peak was only observed in the RREP. The Nf peak may reflect a feed-forward preparation for compensation by a behavioral motor response, simultaneous with respiratory sensory perception. In addition, the Nf peak generated in the prefrontal cortex may be part of the integration of information for motor cortical output as part of the behavioral motor response to loaded breathing (Davenport et al., 1996). The neural generators of the Nf peak, however remain unknown. The cortical projection of skin mechanoreceptors has been identified. Information thansduced by sensory receptors is relayed via the spinal cord to thalamus, then project to specific areas of the cortex. The primary somatosensory cortex has a somatotopic modality-specific organization (Kaas and Pos, 1988). The activation of cortical neurons by mechanical loads has been studied using evoked potential techniques similar to those routinely used in other somatosensory systems (Buchner et al., 1996; Rossini et al., 1996). The P1 peak followed by the N1 peak was identical in all evoked potentials with different latencies and amplitudes. Minor differences in the extent of negative and positive fields on the scalp could be observed in the same subject when changing the stimulus modality, but evoked potential components and localization followed the pattern suggested by Desmedt et al. (1987). The fundamental difference in sensory nerve conduction measured by electrical stimulation and mechanical stimulation is that electrical stimulation activates the distal

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56 axon and bypasses the transduction process of the receptors. Mechanical receptor potentials are graded and sustained depolarizations of the sensory terminals and nerve impulses are generated by this potential (Katz, 1950). Changes in receptor properties of skin mechanoreceptors in neuropathy have been characterized by high mechanical thresholds without significant change in receptor transduction time (Mizobichi et al., 2002). Inspiratory occlusion, air-puff pressure pulses and chest cutaneous stimulation used in the current study include the transduction time for the afferent mechanoreceptors, most likely large-diameter cutaneous afferents (Johansson and Vallbo, 1979b). The RREP and chest SEP may have similar afferent conduction distance, suggesting that time is required when breathing against the inspiratory occlusion to bring the respiratory mechanoreceptors to threshold. Thus, the latency differences in normal subjects between the RREP and mechanically evoked potentials in the current study may be due to the rise time of the mechanical stimulus (inspiratory occlusion), force transfer process within the respiratory system, the receptor transduction time and the conduction in the terminal axon. The transduction time for respiratory mechanoreceptor activation during inspiratory occlusion is unknown. In addition, the SEP elicited by air puff stimulation to the mouth had a longer latency than the SEP elicited by cutaneous stimulation to the chest wall. This difference may be due to the variations in the rise time of the stimulus that alters the rate of mechanical indentation and the transduction process (Pratt and Starr, 1981). The RREP and mouth SEP may have similar afferent sources (upper airway) and sensory modality (pressure), suggesting that time is required when breathing against the inspiratory occlusion to bring the respiratory mechanoreceptors to threshold. Thus, the amplitude differences in normal subjects between the RREP and mechanically evoked

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57 potentials in the current study may be due more mechanoreceptor populations activated or different neural processing for the respiratory afferent information. The SEP following mechanically air puff stimuli to the mouth had a greater amplitude than the SEP recorded following mechanical stimulation on the chest wall. The difference of the amplitude between the air puff stimulation and the cutaneous stimulation may be due to a wider surface area stimulated with air pressure, greater receptor density and/or differences in sensory amplification by second order neural processing system. In addition, Hashimoto et al. (1992) reported that the SEP elicited by air puff stimulation exhibited an increased amplitude and decreased latency with increasing intensity. Thus, the amplitude differences between the air puff and cutaneous stimulation may be due to different stimulus intensity. However, it is difficult to compare the intensity between two different sensory modalities (pressure versus skin indentation). Conclusion In summary, the observation of the evoked potentials elicited by different types of mechanical stimulation demonstrated that: 1)The inspiratory occlusion may activate mechanoreceptors in the mouth, upper airway, lower airway, lung, and respiratory pump muscle; 2) These mechanoreceptors may activate cortical neurons in the somatosensory region of the cerebral cortex; 3) The P1 and N1 peaks of the RREP elicited by interrupting occlusion are analogous to the P1 and N1 peaks of the SEP elicited by air puff or cutaneous mechanical stimulation.; 4) a similar temporal activation sequence for the P1 and N1 peaks was observed for the RREP and the SEPs; 5) The Nf peak was only elicited by inspiratory occlusion; 6) The RREP P1 and N1 peak latencies were longer than the corresponding peaks of the mouth SEP and chest SEP; 7) The P1 and N1 peak amplitudes of the RREP were significantly greater than those of Chest SEP; 8) The N1

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58 peak amplitude of the RREP was significantly greater than the mouth SEP; 9) There were no significant difference between the chest SEP and mouth SEP for the P1 and N1 peaks; 10) The P1 dipole sources were located in the contralateral somatosensory area for the mouth and chest SEPs and bilaterally in the same somatosensory cortical region for the RREP. Thus, it is concluded that the RREP elicited by inspiratory occlusion processes respiratory mechanical information from multiple, bilateral afferent sources. Mechanically elicited SEPs project to similar regions of the somatosensory cortex. Cortical activation elicited by respiratory mechanical loads is evidenced by RREP recordings. The cortical neurons activated by respiratory mechanical loads elicit the RREP similar to the SEP for other somatosensory systems. This suggests that respiratory mechanoreceptor-activated cortical neurons are in the somatosensory cortex and the information is mediated through somatosensory pathways. The location of the mechanoreceptors and neural mechanism involving the respiratory sensation remain uncertain.

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59 Table 3-1 Group mean amplitudes and latencies for P1 and N1 peaks of the RREP, mouth SEP, and chest SEP. P1 C3' P1 C4' N1 C3' N1 C4' Amplitude (V) RREP 1.89 1.40* 1.90 1.23* -3.90 2.01* $ -4.15 2.55* $ Mouth-SEP 0.94 0.76 1.53 0.91 -1.58 1.45 -2.02 1.39 Chest-SEP 0.56 0.62 1.19 1.1 -1.25 2.05 -1.58 2.41 Latency (msec) RREP 63.49 6.53* $ 107.30 11.82* $ Mouth-SEP 39.91 3.71 76.10 6.08 Chest-SEP 31.00 4.55 54.17 2.76 indicates a significant difference between RREP and chest SEP. $ indicates a significant difference RREP and mouth SEP. indicates a significant difference between right and left cortices.

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60 Mouth SEPChest SEPNf RREP -4-2024A1A2-F4 (V)0 0.1 0.2 0.3 0.4 s -4-2024A1A2-F4 (V) -4-2 02 4A1A2-F4(V) 0 100 200 300msec RREPMouth SEPChest SEPNf Figure 3-1. Comparison of the RREP, Mouth SEP, and Chest SEP from frontal electrode (F4). The black line represents the stimulus. The Nf peak was only observed in the RREP elicited by inspiratory occlusion.

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61 3 -3 A1A2-C3' (V) -2 -1 0 2 1 -2 A1A2-C4' (V) -1 0 1 3 2 600 400 0 200 N1 P1 msec Figure 3-2. Comparison of the RREP from right (C3') and left (C4') electrodes. The black line represents the inspiratory occlusion. P1, which is the positive peak of RREP, represented by black arrow; N1, which is the negative peak of RREP, represented by gray arrow.

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62 -2 A1A2-C3' (V) -1 0 1 P1 N1 2 3 -2 -1 A1A2-C4' (V) 0 1 2 0 100 200 msec 300 Figure 3-3. Comparison of the mouth SEP from right (C3') and left (C4') electrodes. The black line represents air-puff stimulus. P1 is the positive peak of the mouth SEP (black arrow). N1is the negative peak of the mouth SEP (gray arrow).

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63 -3 -2 A1A2-C3' (V) -1 0 1 N1 2 P1 -2 -1 A1A2-C4' (V) 0 1 2 -100 0 100 200 300 400 msec Figure 3-4. Comparison of the chest SEP from right (C3') and left (C4') electrodes. The black line represents the cutaneous stimulus. P1 is the positive peak of the chest SEP (black arrow). N1 is the negative peak of the chest SEP (gray arrow).

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64 Amplitude (uV) -8-6-4-20246 * # P1 RREP P1 Mouth SEP P1 Chest SEP N1 RREP N1 Mouth SEP N1 Chest SEP Figure 3-5. Comparison for the amplitudes of the P1 and N1 peaks of the RREP, mouth SEP and chest SEP. The amplitude of the P1 peak of the RREP was significantly greater than the mouth SEP (p < 0.05). The amplitude of the P1 peak of the RREP was significantly greater than the chest SEP (p < 0.05).The amplitude of the N1 peak of the RREP was significantly greater than the chest SEP (p < 0.05). There were no significant difference between the P1 peak of the mouth SEP and chest SEP. There were no significant difference between the N1 peak of the mouth SEP and chest SEP. indicates a significant difference between the RREP and chest SEP amplitude. # indicates a significant difference between the RREP and mouth SEP.

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65 Amplitude (V) -4-2024 * C4-P1 Mouth SEP C3-P1 Mouth SEP C3-N1Mouth SEP C4-P1Chest SEP C3-P1Chest SEP C4-N1Mouth SEP C4-N1 Chest SEP C3-N1Chest SEP Figure 3-6. Comparisons between the C3' and C4' electrode sites for the P1 and N1 peaks of the mouth SEP and chest SEP. The mouth SEP P1 peak was significantly greater amplitude in the C4' electrode site (p < 0.05). The chest SEP P1 peak was significantly greater amplitude in the C4' electrode site (p < 0.05). indicates a significantly different between the C3' and C4' electrode sites.

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CHAPTER 4 RESPIRATORY LOAD PERCEPTION AND RREP WITH INCREASED BACKGROUND RESISTANCE. Introduction The inspiratory airflow is impeded by applying resistive loads during breathing. The breathing pattern during a resistive loaded breath decreases (more negative) mouth pressure as airflow increases, both parameters peak at mid-inspiration. When the magnitude of the resistive load increased, airflow decreases further due to the impedance imposed by the resistive load (Noble et al., 1972). As the resistive load magnitude increases, more negative pressure is needed to overcome the resistance. When the resistive load magnitude reaches a sufficient level, cognitive awareness (detection) of the load occurs. Load detection (LD is studied using techniques to measure the ability of subjects to signal detection of added loads to breathing. The ability of a subject to detect an added load, R, is a function of the ratio of added load to the initial background load (R 0 ), called the Weber-Fechner fraction, R/ R 0 The detection of resistive loads is dependent of the background resistance of the subjects airways and the breathing apparatus. The detection threshold is defined as the R that is signaled as detected for 50 % of the load presentation, R 50. The threshold for detection of resistive loads has been reported to be a constant fraction R 50 / R 0 of about 0.3 for normal subjects and patients with respiratory disease. The perception of respiratory mechanical events depends on cognitive processing of afferent information from mechanoreceptors in the respiratory system. Bennett et al. (1962) reported a variable detection threshold R 50 for resistive 66

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67 loads in healthy adults of 2-4 cmH2O//sec. Wiley and Zechman (1966) reported that the relationship between the background resistance and the detection threshold for a various background load conditions was 0.25-0.3 despite a large variability in the R detection threshold values. The variability in the detection threshold value among these studies was thus due to the variation in the background resistance, which includes both the subjects intrinsic respiratory resistance and the resistance of the apparatus. Killian et al. (1980) compared the detection threshold when the resistive loads were applied at different inspiratory flows with different background resistances. They suggested that the resistive load was modulated by the relationship between pressure and airflow over the early part of inspiration, rather than changes of pressure alone. Zechman et al. (1985) reported that transdiaphragmatic pressure (Pdi) increased significantly just before detection of added loads without change in airflow or volume. They concluded that load detection occurred when the change in Pdi reached a critical level. This study demonstrated that there was a transdiaphragmatic threshold for load detection and implicated that the respiratory pump may be a source for the afferent signals of load detection (Zechman et al., 1985). The pattern of airflow, volume and pressure changed by adding resistive loads and the perception of the added resistive load was also changed by adding the background load (Revelette et al., 1990). These mechanical changes may be mediated by afferents from the mouth, airways and respiratory muscles. Multiple respiratory sensory mechanisms may contribute to the respiratory sensation when breathing against a mechanical load. Breathing against mechanical loads may induce changes in respiratory

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68 mechanics and there must be pathways to mediate afferent information projection from respiratory mechanoreceptors to reach the cortex. Poor perception of respiratory loads in patients with chronic pulmonary disease may be due to altered processing of afferent information from respiratory mechanoreceptors. It has been reported that the perceived load magnitude declines with a background resistive load of 14.2 cmH2O (Burdon et al., 1983). Decreased magnitude perception of inspiratory resistive loads occurred when a higher background load was present (McCloskey, 1973). Thus, it is likely that load detection may only require the activation of the sensory pathway while magnitude estimation may involve evaluation of information from multiple afferent inputs. The short-latency components of evoked potentials reflect the integrity of the primary sensory pathways and vary with stimulus parameters such as intensity and frequency (Nagamine et al., 1998). The long-latency components of evoked potentials reflect cognitive and psychological state of the subjects (Webster and Colrain, 2000). Inspiratory occlusion produces a series of short-latency RREP peaks, P1 and N1 peaks recorded from electrodes overlying the somatosensory cortex and Nf identified over the frontal region of the cortex (Davenport et al., 1996; Webster and Colrain, 2000). Inspiratory occlusion of the respiratory system elicited the RREP reflecting the activation of respiratory mechanoreceptors (Davenport et al., 1986). Dipole sources for the P1 and Nf peaks were bilaterally located within primary somatosensory and supplementary motor cortices, respectively (Logie et al., 1998). However, the pathway of respiratory loaded information to the cortex is unknown

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69 Knafelc and Davenport (1997) demonstrated a relationship between the amplitude of the P1 peak of the RREP and the perceived magnitude of inspiratory resistive loads. They reported that the P1 peak amplitude of the RREP was a function of the perceived load and proportional to the magnitude of the resistive load. However, the relationship between the effects of increased background resistance on the RREP is known. If the RREP is associated with perception of added loads then the addition of an increased background resistance should affect the RREP. Therefore, the purpose of the present study is to determine the effects of background load on RREP responses to inspiratory resistive loads. It was hypothesized that short-latency components (P1, N1, and Nf) of the RREP are involved in conscious perception of respiratory mechanical loads and are recorded only when the added resistive load is above the detection threshold of the subject. It was further hypothesized that a resistive load detectable without increased background resistance may have an RREP but the RREP may not be present when the background is increased to make the same load undetectable. Method Subject Subjects (10 men, 10 women, averaged age= 25.8 yr) with no history of cardiovascular disease, neurological disease, no history of smoking and no evidence of current major illness participated in the study. The Institutional Review Board of University of Florida reviewed and approved the protocols used in this study. All subjects were informed of the nature of the study before starting the experiment and written consent was obtained.

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70 Pulmonary Function Testing All subjects were seated upright in a chair to perform a pulmonary functional test that conformed to American Thoracic Society Standards. The subjects must have an FEV1 greater than 70% predicted to continue in the study. Procedures The subject was seated comfortably in a lounge chair in a sound-isolated chamber, separated from the experimenter and experimental apparatus. A standard set of instructions was presented to the subjects to inform them of their task. The subject respired through a mouthpiece and nonrebreathing valve (Hans Rudolph, 2600 series, Kansas City, MO) connected to a loading manifold. The loading manifold was hidden from the subjects view and connected to a pneumotachograph (Hans Rudolph, Model 4813) attached by reinforced tubing to the inspiratory port of the nonrebreathing valve. The resistive loads were sintered bronze disks placed in series in the loading manifold and separated by stoppered ports. Mouth pressure (P m ) was recorded from a port in the center of the nonrebreathing valve. The P m was displayed on an oscilloscope for timing the load applications and stored on a computer (Model 1401, Cambridge Electronic Design). An electrode cap with integral electrodes was used to record scalp EEG activity. The electrode positions were based on the International 10-20 System. The cap was placed on the subjects head, positioned and secured with a strap. Scalp and electrode contact was made by the application of electro-conducing paste administered through the center opening in the electrode. The impedance levels for each electrode was checked and maintained below 5 K. The electrode cap was then connected to an EEG system (Model 12, Grass instruments, Quincy, MA). EEG activity was monitored with an oscilloscope

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71 monitor. The EEG activities were recorded and referenced to the joined earlobes. Two electrodes were placed over the lateral edge of the eye for recording vertical electrooculograms (EOG). Any load presentation that occurred with an eye-blink was discarded. The EEG signals were band-pass filtered (0.3 1 KHz), amplified and sampled (2 kHz) for computer analysis. The EEG activities were led into signal averaging computer system (Model 1401, Cambridge Electronics Design). All subjects were instructed to relax all postural and facial muscles and to breathe as normally as possible. The subject wore a nose clip and breathed through a mouthpiece connected to the non-rebreathing valve and loading apparatus. The subject inspired through the minimum resistance port of the loading manifold. The occlusion valve was connected to the opening of this minimum resistance port. The inspiratory load was presented by silently inflating the occlusion valve at approximately the mid-inspiratory point of the breath. Activation of the occlusion valve closed this port and channeled the inspired air through one of three manifold resistance ports. When a resistance was presented, the stopper for that port was removed. Activation of the occlusion valve on the minimal resistance port was then closed and the subject inspired through the increased resistance. The inspiratory resistive load was presented as an interruption of inspiration. The balloon pressure was used to trigger the computer for data sample collection. Each load was separated by 2-6 unloaded breaths. The load was applied for a duration of 500 ms. Previous results from this laboratory have shown that the averaged detection threshold value for resistive loads in healthy subjects with no background load is 1-2 cmH 2 O//s. One resistive load that was below the detection threshold and two resistive loads that exceed the

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72 detection threshold were used for eliciting the evoked potentials (R1=0.2, R2=3.8, and R3= 23.3 cmH2O//s). Two trials were presented: 1) without elevated background resistance and 2) with an elevated background resistance of 13.3 cmH2O//s. Each trial was separated into two sets of load presentations. Each set was separated by a rest period off the breathing apparatus. The order of the presentation of the three resistive loads was randomized to minimize temporal, order and sequence effects. Each set consisted of 120 presentations. Within each set of 120 load presentations were tested by using 24 blocks of five presentations of each load. Ordering of the different presentation blocks was randomized, with the restriction that each successive set of 3 blocks contained only one block of each resistive load level. Thus, over the set of 120 presentations each block of five trials of a given load was presented 8 times (40 total presentation of each load magnitude). The randomization was independently drawn for each set of 120 trials and for each subject with the 2 sets of presentations. Thus, each load magnitude was presented 80 times. The time required to present the entire study was ~3 hours, including subject preparation time. For 5 of the 20 subjects, an additional experiment was performed with inspiratory occlusion replacing R3. Data Analysis The Pm and EEG activity for each load presentation were digitized and stored on the computer for subsequent signal averaging (Signal 2, Cambridge Electronics Design). Each resistive load magnitude was averaged separately. The data analysis was performed for each load magnitude including the loads from the two trials. Individual subject files were used for RREP peak component identification, amplitude and latency analysis. Baseline-to-peak amplitude values were determined separately for each peak. Peak amplitudes were correlated with the resistive load

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73 magnitudes. The P1 peak is the positive potential occurring within a 40-70 ms time window. The N1 peak is the negative potential occurring between 80-120 ms. The Nf peak is the negative peak in the frontal electrodes occurring at 25-40 ms. The latency and amplitude of each RREP peaks (P1, N1 and Nf) were compared using one-way, repeated-measures analysis of variance (ANOVA). A p < 0.05 was considered as statistically significant. Results P1, N1, and Nf peaks were present in all subjects with no-background resistance for R2 and R3 loads (Fig 4-1a and Fig 4-1b). With increased background resistance, P1, N1 and Nf peaks were present in all subjects with the increased background load only for the R3 load and inspiratory occlusion (Fig 4-2a and Fig 4-2b). There was no significant effect of background resistance for the latencies of the P1, N1, and Nf peaks of the RREP for each inspiratory resistive load, and occlusion when present (Table 4-1). Mean amplitudes of the P1, N1 and Nf peaks for each inspiratory resistive load and occlusion are presented in Table 4-1. P1 and Nf peaks showed a significant main effect of resistive load magnitudes with no-background resistance. The P1 peak amplitude showed a significant difference between no-background resistance condition and increased background resistance condition (Fig 4-3). The R 50 remained the same but R 0 changed to R 0 when increasing the background resistance. Thus, the baseline background resistance was R 0 = 4.8 cmH2O//s, averaged resistance of the subject and apparatus with no-background load. The background resistance was increased to R 0 '= 16.4 cmH2O//s, the resistance consisted of the subject and 13.3 cmH2O//s increased background resistance. The ratio (R 50 /R 0 ) is 0.3 for normal subjects. R2/R 0 is changed with the increased background

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74 resistance increased to 0.1 and becomes undetectable. There was no P1 peak present with R2. R3/R 0 was not changed with the background resistance increased and was detectable as it was above 0.3. Thus, P1 peak amplitude of the RREP is the function of the detection threshold (Fig 4-4). Discussion The RREP peaks, P1, N1, and Nf were present with detectable resistive load magnitudes (R2 and R3) and were not present with the load (R1) below the detection threshold for the no-background resistance trials. In contrast, P1, N1, and Nf peaks were absent with R1 and R2 loads below the detection threshold with increased background. All the peaks of the RREP were present with R3 load, which was above the detection threshold with increased background resistance. These results demonstrated that the P1, N1 and Nf peaks are related to load perception and changes in perception changes the RREP peaks. Inspiratory resistive loads require the inspiratory muscles to overcome the load to generate airflow (Wiley and Zechman, 1966). The increased tension developed by the inspiratory muscles may be the stimulus mediating the perceived magnitude of added loads (Killian et al., 1980; Killian et al., 1984). Resistive loads are airflow-dependent loads and a decrease in the inspiratory airflow reduces the pressure changes associated with the increased load. The decrease in these load-related mechanical parameters with a decreased inspiratory effort can result in a reduced sensory stimulation and underestimation of the load magnitude. The ventilatory response to external mechanical loads depends on the type of load (Younes et al., 1994). It has been reported that both minute ventilation and mean inspiratory flow rate decreased when breathing against a resistive load in conscious man (Dressendorfer et al., 1977). The changes in the breathing

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75 pattern compensate for the increased work of breathing (Zechman et al., 1985). However, the afferent mechanisms involved in the behavioral load compensation responses remain unclear. When a stimulus of constant intensity is applied, the sensation it produces has two stages. The first stage consists of an increased sensory magnitude; during the second stage the sensation diminishes in magnitude over time as accommodation produces adaptation to the load. The afferent pathway mediating the load detection has not been identified; however, the receptors within the lung, chest wall, and upper airways may be involved in respiratory sensation (Davenport et al., 1991; Knafelc and Davenport, 1997; Harver, 1987). The detection of an inspiratory load is a complex chain of events involving cortical sensory processing of respiratory mechanoreceptor activity (Kifle et al., 1997; Fritz et al., 1999). Burdon et al. (1983) reported that perceptual performance was not altered by increased background resistance. It is likely they failed to demonstrate an effect of increased background load in scaling performance because their protocols resulted in decreased airflow and loss of stimuli with increased background resistance. Revelette et al. (1984) reported that the shift in the exponent for magnitude estimation with increased background resistance is associated with an increase in the load detection threshold. This is consistent with our findings in the present study that the resistive load, below the detection threshold with the increased background load will not elicit RREP. The activation of cortical neural activity by resistive loads has been studied using evoked potential techniques (Knafelc and Davenport, 1997). The RREP elicited by inspiratory occlusion applied at mid-inspiration was recorded from the somatosensory region of the cortex in healthy adults (Davenport and Revelette, 1990; Davenport et al.,

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76 1986). Early peaks of the RREP were identified in healthy adults, asthmatics and double lung transplant recipients (Davenport et al., 1996; Kifle et al., 2000; Zhao et al., 2003). The P1 peak reflects the depolarization of cortical neurons by the arrival of afferent activity stimulated by the occluded breath. Franzn and Offenloch (1969) reported that the magnitude of the early positive peak of the touch elicited somatosensory evoked potential (SEP) was correlated with mechanical stimulus amplitude and magnitude estimation of the touch. If the RREP is analogous to the SEP, then the amplitude of the P1 peak of the RREP should correlate with the load magnitude. This is consistent with the result from Knafelc and Davenport (1997) reporting a relationship between the RREP P1 peak amplitude and resistive load magnitude. They reported that there was a log-log relationship between the magnitude of the resistive load and P1 peak amplitude when a series of resistive loads were applied (Knafelc and Davenport, 1997, 1999). However, it remained unknown if the threshold for eliciting the RREP corresponded to the detection threshold. The relationship between the RREP P1 peak amplitude and the resistive load magnitude was found in the present study that the relative afferent signal strength would be changed by increasing the background resistance. Thus, the RREP peaks were present or absent depending on the detectability of the load. If the load was above the detection threshold, the RREP was present. However, if the load was below the detection threshold, the RREP was absent. Moreover, changing the detection threshold by adding the background resistance decreased the amplitude of the load that remained detectable and the RREP was not present for the load forced below the detection threshold by the elevated R 0 This further supports the conclusion that the amplitudes of RREP peaks are directly related to the detectability and

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77 magnitude of the load. RREP elicited by inspiratory occlusion was found unaffected by increased background resistance in the present study. This result was consistent with previous finding that the RREP elicited by the inspiratory occlusion remains unchanged with different type of loads and different background environments (Davenport et al., 1981). Variations in RREP latencies and amplitudes in the current study provided evidence that cortical processes to mechanical loads were affected not only by the magnitude of load but also by the detection of the loaded information, suggesting a gated respiratory afferent feedback system. In the present study, the P1 peak amplitude of the RREP is not present with the increased background resistance because the applied resistive load is below the detection threshold ratio. The fact that the RREP is not present with increased background resistance suggests that the respiratory resistive load information was gated out by changing the background ventilatory state, such as increased background resistance. Thus, the respiratory afferent information will be gated in if above the detection threshold or will be gated out if the threshold was unregulated by other systems, such as increased background resistance. Although the interaction between the conscious and the automatic control of breathing is uncertain, failure to have sufficient respiratory motor drive to meet the ventilatory demands will result in a conscious awareness of breathing and behavioral motor response. Thus, there must be a subcortical threshold to gate-in or gate-out the respiratory afferent input and feedback motor output. However, neural indicator and the possible mechanisms that subserve the respiratory sensory gating system are unknown.

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78 Conclusion In summary, the present study demonstrated that activation of cortical neural activity was associated with the perceptual processing of resistive load information. The detection threshold of inspiratory loads was increased by the increased background resistance but the relative threshold R 50 /R 0 is unchanged. If the resistive load was detectable, the RREP was present. If the load was not detectable or becomes undetectable with increased background load, the RREP was not present, supporting the hypothesis that the RREP response could be changed by increasing the background load which shifts the detection threshold. Therefore, perceptual processing of respiratory load information may be gated-in subcortical centers as part of the central afferent pathway. Future studies are necessary to investigate the respiratory patterns and the cognitive component of the RREP P300 peak to extend these studies to the gating effects of the cognitive measures of respiratory mechanoreceptor processing.

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79 Table 4-1 Group-averaged P1, N1, and Nf latencies and amplitudes of RREP for inspiratory resistive load and inspiratory occlusion. P1 C3' P1 C4' N1 C3' N1 C4' Nf F3 Nf F4 Amplitude (V) R2 No Background 1.40 0.69 1.21 0.53 -2.30 0.85 -2.38 0.90 -1.77 0.98 -1.71 0.62 R3 No Background 2.20 0.73 # 2.29 0.57 # -2.85 1.04 -3.14 1.41 -2.27 0.78 # -2.32 0.85 # Occlusion No Background 2.31 0.93 2.38 0.97 -2.02 0.39 -2.31 0.65 -2.09 0.80 -2.11 0.20 R3 With Background 1.79 0.75 1.79 0.74 -2.45 0.88 -2.54 0.95 -1.66 0.97 -1.70 0.81 Occlusion With Background 1.85 0.22 2.12 0.67 -3.01 0.93 -3.13 1.00 -1.69 0.40 -1.72 0.32 Latency (msec) R2 No Background 65.73 7.19 104.74 8.99 34.88 5.64 R3 No Background 63.57 6.70 100.54 10.05 36.75 5.84 Occlusion No Background 64.53 4.61 104.61 7.50 34.05 5.61 R3 With Background 72.20 8.40 109.60 13.52 35.30 3.76 Occlusion With Background 69.98 9.02 105.09 10.24 31.28 6.37 Values were means SD. Occlusion was an infinitive load (maximal load) for the respiratory system. # indicated the significant difference between R2 and R3 resistive loads. indicated the significant difference between no-background resistance and increased background resistance.

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80 (a) No background resistance R1 10 V Nf R2 + R3 N f 0.4 sec 0 0.2 0.6 0.8 (b) With background resistance R1 10 V + R2 Nf 0 0.2 0.4 sec 0.6 0.8 Figure 4-1. Nf peaks recorded from F4 electrode with different resistive loads in both no-background resistance (a) and increased background resistance (b).

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81 (a) No background resistance + 10 V N1 P1 N1 P1 R1 R2 R3 0.2 0.4 0.6 0 0.8 sec (b) With background resistance + N1 P1 10 V R2 R3 R1 0.2 0.4 0.6 0.8 0 sec Figure 4-2. P1 and N1 peaks recorded from C4' electrode with different resistive load in both no-background resistance (a) and increased background resistance (b).

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82 RREP peak response to load11.522.533.5 load magnitudeamplitude P1-no load P1-with load N1no load N1-with load Nf-no load Nf-with load Figure 4-3. P1, N1 and Nf peaks recorded from C4' and F4 electrode sites, respectively with inspiratory occlusion (maximal resistive load) in both no-background resistance and increased background resistance conditions.

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83 P100.511.522.5012345dR/R0P1 ampltude R3 No background R2 No-background R3 with-background R2 with-background 0.3 Figure 4-4. Relationship between amplitude of P1 peak and changes of resistive load magnitude with different background resistance. The change in resistive load magnitude was determined by resistive load magnitude divided by baseline resistance (R 50 /R 0 ). R 0 = 4.8 cmH2O//s was the baseline resistance measured from both subject and apparatus with no-background load. R 0 '= 16.4 cmH2O//s is the baseline resistance measured from both subject and apparatus with increased background load. The ratio (R 50 /R 0 ) is 0.3 for normal subject. R2 became undetectable as it was below 0.3 after increasing the background resistance. Therefore, there was no P1 peak present when R2 load was applied with increased background resistance.

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CHAPTER 5 GENERAL DISCUSSION Summary of Results 1. The results of the evoked potentials elicited by inspiratory occlusion and cutaneous stimulation demonstrated that short-latency components of the RREP and SEP project to the same scalp locations. The RREP components were observed bilaterally whereas the SEP was observed contralateral to the stimulated hand. The scalp distributions of the RREP and SEP P1 peaks were found at the centroparietal (C4') electrode position. The latencies of the P1 and N1 peaks of the RREP were significantly longer than the SEP. The amplitudes of the P1 and N1 peaks of the RREP were significantly greater than the SEP. The Nf peak was only observed in the frontal region of RREP. 2. The P1 and N1 peaks of the RREP elicited by inspiratory occlusion were similar to the peaks of the SEP elicited either by air puff stimulation to the mouth or cutaneous mechanical stimulation to the chest. The Nf peak was only observed in the frontal sites of RREP. The latencies of the RREP peaks were longer than the mouth SEP and chest SEP. The P1 and N1 peak amplitudes of the RREP were significantly greater than the chest SEP. The N1 peak amplitude of the RREP was significantly greater than the N1 peak of the mouth SEP. There was no significant difference between chest SEP and mouth SEP for P1 and N1 peaks. 3. The RREP response to resistive load magnitude was correlated with the presence of the RREP P1, N1, and Nf peaks with detectable resistive loads. The RREP was not 84

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85 present with loads that were below the detection threshold. When an increased background resistance was applied, the RREP peaks were present with the load that was above the detection threshold but were absent with the load that was below the detection threshold. Distribution of Evoked Potentials RREP with Inspiratory Occlusion RREP peaks, P1, N1 and Nf were identified in response to inspiratory occlusion, consistent with previous studies (Davenport et al., 1996; Davenport et al., 2000; Davenport et al., 1986). The amplitude of the P1 peak of the RREP increased with the magnitude estimation of the corresponding load and the inspiratory load magnitude (Knafelc and Davenport, 1997; 1999). In the present study, these RREP peaks were recorded in all three experiments. The RREP was not present however, if a load was below the load detection threshold. The P1, Nf and N1 peaks of the RREP were found bilaterally. The P1 peak location is consistent with the dipole in the somatosenory cortex. The Nf peak location is consistent with the dipole in the supplementary motor cortex. The N1 peak location is consistent with the midline of the cortex, greatest at the vertex. SEP with Mechanical Stimulation The P1 and N1 peaks of the limb SEP, mouth SEP and chest SEP were identified in scalp locations that were contralateral to the side stimulated. These peaks were not bilaterally symmetrical, a difference from the RREP. The electrode sites of the P1 peak of the limb SEP, mouth SEP and chest SEP were over the post-central gyrus lateral to the midline which is consistent with the somatotopical representation of the fingers in the primary somatosensory cortex (Pratt and Starr, 1981). Thus, the mechanical SEP and the RREP P1 peaks are consistent with an activation of neurons in the primary

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86 somatosensory region of the cerebral cortex. The electrode sites of the N1 peak of the limb SEP, mouth SEP and chest SEP were over the central sulcus at the midline of the cerebral cortex which is also consistent with the somatotopical representation of the fingers in the sensorimotor cortex (Pratt and Starr, 1981). Thus, the mechanical SEP and the RREP N1 peaks are consistent with an activation of neurons in the sensorimotor region of the cerebral cortex. Mechanical stimulation activates mechanoreceptors in a defined focal location in the hand, chest or buccal surface of the mouth. The mechanoreceptors are myelinated afferents with a rapid conduction velocity. The latency for mechanical cutaneous stimulation included mechanical transduction time and pathway conduction time. The latency for skin (limb and chest) mechanoreceptors to the cortex is less than either the mouth or RREP. However, the cutaneous peak amplitudes were smaller, most likely due to fewer mechanoreceptors activated by the focal stimulation compared to the widely distributed stimulation with inspiratory occlusion. Latency and Amplitude of Short-Latency Peaks of Evoked Potential RREP The P1 peak of the RREP has been reported to reflect the arrival of respiratory mechanoreceptor activity in the primary sensory area of the cortex (Logie et al., 1998). The P1 peak is determined mainly by the physical characteristics of the stimulus. The RREP P1 peak amplitude was correlated to the resistive load magnitude (Knafelc and Davenport, 1997; Webster and Colrain, 2000), suggesting that this peak is the indicator of the stimulus dependent activation of somatosensory cortical columns. The amplitude of RREP N1 peak was reported to decrease with a decrease of the load magnitude (Bloch-Salisbury et al., 1998). The P1 and Nf peak amplitudes were unaffected by attention

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87 while attention increased the N1 amplitude (Harver et al., 1995). Zhao et al. (2002) also reported that there were no significant difference in either latency or amplitude of the P1 and Nf peaks of the RREP peaks between attend trials and ignore trials for both double lung transplant recipients and matched healthy subjects. Thus, the P1 and Nf peaks are dipoles in the cerebral cortex indicating the arrival and magnitude of respiratory afferent activation. It is unlikely that the frontal Nf peak of the RREP reflects myogenic activity. The neural generators of the Nf peak are consistent with a pre-motor cortical location. The bilateral distribution of the Nf peak and the latency suggest that it reflects a parallel (to P1) input to activity in prefrontal structures. It is generally recognized that thalamocortical inputs to the somatosensory cortex project predominantly to deep cortical layers and that they generate a surface (P1) positive potential (Wood and Allison, 1981; Wood et al., 1985). The frontal Nf peak was not preceded by the P1 peak and can not be explained as voltage spread from the somatosensory cortex. The negative peak suggest a dipole generator in the more superficial layers of the prefrontal cortex The prefrontal cortex gated the early stage of somatosensory processing and can exert an inhibitory modulation on sensory processing (Yamaguchi and Knight, 1990). The prefrontal cortex mediates cortical motor reflexes suggesting that the generation of the frontal Nf potential is a pre-motor respiratory cortical response to the respiratory load. The Nf peak generated in the prefrontal cortex may therefore function as the integration of load information related to the behavioral motor response to the mechanical stimulus produced by loaded breathing (Davenport et al., 1996). However, the exactly source of the Nf peak is unknown.

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88 The N1 peak of the RREP was the negative peak associated with the P1 peak of the RREP (Davenport et al., 1986). The N1 peak of the RREP was observed to be of greatest amplitude in the central electrode sites. These electrode sites were over the central sulcus at the midline of the cerebral cortex which is consistent with the somatotopical representation of the fingers in the sensorimotor cortex (Pratt and Starr, 1981). In addition, Webster and Colrain (2000) reported N1 peak is recorded from the vertical dipole source and is affected by the attention. Thus, the N1 peak of RREP may be the second order cortical processing of respiratory afferent information. Bloch-Salisbury et al. (1998) reported that N1 peak amplitude of the RREP decreases as the resistive load decreased, similar to P1 peak of the RREP. Thus, the N1 peak of the RREP may be the second order cortical processing of the respiratory afferent information that is based on perceiving the first-order somatosensory information and may affect the cognitive processing. SEP on Hand The P1 peak was elicited by mechanical cutaneous stimulation of the load in the C3' and C4' electrode sites in the present study. This is consistent with the reports of SEP elicited by mechanical stimulation of the digits (Onofrj et al, 1990; Pratt and Starr, 1981), suggesting that the generation of this early positive peak was in the primary somatosensory region of the cortex (Hamalainen et al., 1990). In the current study, the latency of the SEP P1 peak elicited by cutaneous stimulation of the hand was about 25 msec. This is consistent with the report of Schieppati and Ducati (1984) that the P1 peak of the SEP is elicited by the cutaneous stimulation. Thus, the results of the present study are consistent with the P1 peak of the SEP generated in the somatosensory region of the cortex.

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89 The P1 peak of the SEP generated by mechanical stimulation have been found in area 3b (somatosensory cortex) mediated by thalamocortical afferents from ventroposterolateral nuclei in the thalamus and in area 3a activated by afferents from ventroposterior nuclei in the thalamus (Dieter et al., 1986). Neurophysiological studies (Hashimoto et al., 1991; Johansson et al., 1980) that utilize the EEG as indices of sensory processing showed increased evoked potential P1 peak amplitudes were generally produced by an increase in the stimulus intensity. The P1 peak of the evoked potential in response to mechanical stimulation has been identified in the somatosensory regions of the cortex (Hamalainen, 1990). Thus, the P1 peak amplitudes of both the RREP and SEP are a function of the magnitude of neural activity directly correlated with the magnitude of stimulus. SEP on Chest and Mouth The activation of cortical neurons by mechanical loads has been studied using evoked potential techniques similar to those routinely used in other somatosensory systems (Buchner et al., 1996; Rossini et al., 1996). In the present study, minor differences in the extent of negative and positive fields on the scalp could be observed in the same subject when changing the stimulus modality, but the sequence of evoked potential peaks and scalp distributions followed the same pattern regardless of the stimulus modality. Air-puff stimulation selectively activated cutaneous mechanoreceptors (Schieppati and Ducati, 1984). Variations in the rise time of the stimulus can alter the rate of mechanical indentation of the chest wall and the transduction process of the afferent information. The onset of the first SEP peak elicited by mechanical stimulation has been delayed 2-4 ms from that of the SEP elicited by electrical stimulation (Pratt and Starr,

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90 1981). The latency difference was mainly the result of receptor transduction time and mechanical threshold differences from varies receptor locations (Mizobuchi et al., 2000). The SEP following mechanical air puff stimuli to the mouth had a greater amplitude than the SEP recorded following mechanical stimulation on the chest wall. The difference of the amplitude between the air puff stimulation and the cutaneous stimulation may be due to a wide surface area stimulated with air pressure, greater receptor density and/or differences in sensory amplification by second order neural processing system. The RREP and mouth SEP may have similar afferent sources (upper airway) and sensory modality (pressure), suggesting that time is required when breathing against the inspiratory occlusion to bring the respiratory mechanoreceptors to threshold. Thus, the amplitude differences in normal subjects between the RREP and mechanically evoked potentials in the current study may be due huge number of mechanoreceptor populations activated or different neural processing for the respiratory afferent information. Afferent Mechanisms of Evoked Potentials Sources of RREP The P1 peak of the RREP is believed to reflect the arrival of respiratory afferent information in the primary somatosensory region of the cortex. Therefore, the application of inspiratory occlusion, which puts an infinite resistive load on the entire respiratory system, will bilaterally stimulate multiple respiratory mechanoreceptors mediating the neural activity in the cortex. Although the stimulated afferents are unknown, it is likely that mechanoreceptors in the respiratory pump and airways are the primary source of load related somatosensory activity. It has been reported that RREP peaks are present in subjects with denervation of lung vagal afferents (Zhao et al., 2002; 2003). This

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91 laboratory has also reported the presence of the P1, Nf N1 and P300 RREP peaks in double lung transplant recipients respiring through their tracheal stoma, which dennervates both lung vagal afferent and upper airway afferent inputs (Davenport et al, abstract, 2004). Thus, respiratory pump afferents can elicit the RREP with inspiratory occlusion. However, inclusion of mouth, pharyngeal and laryngeal afferents increases the amplitude of the RREP peaks, suggesting the RREP in intact subjects is the result of multiple respiratory mechanoreceptor modalities. Afferent Pathways of RREP and SEP The waveform of the RREP is similar to cutaneous SEPs in the present study. Inspiratory occlusion can alter the afferent activity of several populations of mechanoreceptors in the mouth, pharynx, larynx, lung and respiratory muscles. Inspiratory occlusion halts the normal changes in inspiratory airflow and lung volume. Animal studies have demonstrated that inspiratory resistive loads activate the proprioceptors of respiratory muscles (Jammes et al., 1986). Recordings of cortical responses to afferent stimuli confirm that the electrical responses at 50 ms and 100 ms latencies are generated by activity in SI and SII somatosensory cortex (Hari, 1980). The SEP evoked by electrical stimulation of the median nerve at the wrist or the index finger also showed a similar SEP distribution (Onofrj et al., 1990; Grisolia and Wiederholt, 1980). The results of recordings from the exposed cortex in humans suggest that the short latency peaks of the SEP were generated from the primary somatosensory cortex (Allison et al., 1989a). The results from current SEP study with cutaneous stimulation of hand were constant with previous studies (Johansson et al., 1980; Gardner et la., 1984; Hashimoto et al., 1990), in which the latency delays for mechanical stimulation were accounted for by two peripheral

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92 mechanisms: skin indentation and receptor transudation and lower conduction velocity of cutaneous afferents. Comparisons between RREP and SEP Evoked potentials elicited by mechanical stimulation of skin is affected by respiratory phase (Balzamo et al., 1995; 1999) however, no study has compared the cortical processing of skin and respiratory mechanical stimulation. In the present study, the latencies and amplitudes of the SEP elicited by mechanical air puff stimulation of the mouth and cutaneous stimulation of the hand and the chest wall were compared to the RREP components elicited by inspiratory occlusion. Results from this study showed that the mechanically loaded inspiratory occlusion activates cortical neurons in the somatosensory cortex eliciting peaks analogous to the SEP P1 and N1 peaks. The P1 and N1 RREP peaks had longer latencies and greater amplitudes than the corresponding SEP peaks. The difference in amplitude may be due to the greater activation of mechanoreceptors in the respiratory system. The difference in latency may be due to longer time required to recruit respiratory mechanoreceptors and bring them to threshold. The Nf peak was only elicited by inspiratory occlusion and is a unique feature of the RREP. Knafelc and Davenport (1997) demonstrated a relationship between P1 peak amplitude and the magnitude of resistive loads. They reported that the amplitude of the P1 peak of the RREP significantly increased with increased resistive load magnitude. If the RREP is analogous to the SEP, then the amplitude of the P1 peak of the RREP should be correlated with the load magnitude. This is consistent with the report from Franzn and Offenloch (1969) that the magnitude of the touch elicited P1 peak of the SEP was correlated with mechanical stimulus amplitude and magnitude estimation of the touch.

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93 Thus, the location, origin and amplitude of the RREP peaks correlate with the analogous peaks of the SEP. The Relationship between RREP and Load Perceptual Process Relationship between RREP and Load Perception The detection threshold for resistive loads reported by Bennett et al. (1962) and Wiley and Zechman (1966) resulted in a Weber ratio of approximately 0.3. The detection threshold is a fraction of the background resistance. Variability between subjects when presenting the R2 (3.8cmH2O/l/s) load is likely due to the variability of their intrinsic resistance. Burdon et al. (1983) reported that magnitude estimation is not altered by increased background load. It is likely that they failed to demonstrate an effect of increased background load in their scaling task because their protocols resulted in the loss of lower stimuli with increased background resistance. The finding in the present study that resistive loads below the detection threshold do not elicit the RREP is consistent with the relationship between cortical activation and load detection. If a load does not elicit somatosensory cortical activity then detection of the load does not occur. Thus, the load must be above the Weber ratio detection threshold for the RREP to be elicited. Knafelc and Davenport (1997) demonstrated a close relationship between the RREP P1 peak amplitude and magnitude estimation. They reported that there was a linear relationship between magnitude estimation of the resistive load and P1 amplitude. Similarly, there was a relationship between changes of RREP P1 peak amplitude and the load magnitude in the present study that is relative to the afferent signal strength above the increased background resistance. The RREP peaks were present or absent depending on the detection threshold. If the load was above the detection threshold, then the RREP was present and the P1 peak amplitude was a function of the load magnitude. The

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94 detection threshold can be changed by adding background resistance (Revelette et al., 1984). The RREP was not elicited with increased background resistance that made the resistive load below the detection threshold. The RREP was elicited without the background resistance because the R 50 was above the detection threshold. Thus, a load that elicited the RREP was made undetectable with increased background resistance and the RREP could no longer be observed. RREP Responses to Different Load Magnitude Inspiratory airflow is impeded by applying a resistive load during breathing and the breathing pattern is changed during a loaded breath (Davenport et al., 1992). Thus, when the magnitude of the resistive load increased, airflow tended to decrease due to the impedance imposed by the resistive load. As the load increased, more negative pressure was needed to overcome the resistance. The peak negative pressure occurred at about mid-inspiration when airflow was highest. The pressure and airflow relationship was changed by increased background resistance. Variations in RREP latencies and amplitudes found in current study suggest that cortical responses to mechanical loads are affected not only by the magnitude of load but also by the detection of the load and may be mediated by a gated feedback afferent system. Short-Latency RREP Peak in Response to Increased Background Resistance Wiley and Zechman (1966) studied the relationship between the detection threshold and the background resistance. They have reported variable detection thresholds but a constant Webers Ratio (0.3) with different background resistances. Burdon et al. (1983) reported that increased background resistance was associated with a change in the exponent for magnitude estimation of the resistive load. In contrast, Revelette et al. (1984) reported a significant increase in the power function for load magnitude

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95 estimation with increased background resistance. In the present study, the RREP was present only if the applied resistive load was greater enough to be perceived. Many studies have examined the relationship between the perceived magnitude of loads and the intensity of the loads (Burki et al., 1983; Gottfried et al., 1981; Killian et al., 1981; Wolkove et al., 1981). The present study was the first to compare the RREP response to load perception with increased background resistance. The result from the present study showed that the P1 peak of the RREP was present if the load could be perceived and the amplitude of the P1 peak was larger if the perceived load magnitude was increased. This finding is consistent with the report of Knafelc and Davenport (1997). They demonstrated the relationship between the P1 peak amplitude of the RREP and the perceived magnitude of loads, the amplitudes of the RREP P1 peak was a function of perceived load magnitude and proportional to the intensity of resistive load. With prolonged increases in background resistance, the perceptual sensitivity in healthy subjects is unlikely to be changed from normal and any adjustment in ventilation made on the basis of sensory perception would be expected to be maintained if the load is detected (Killian et al., 1984). This is important clinically to those patients with chronic pulmonary disease. These patients have the greatest need for optimizing their breathing strategy of respiratory muscle force production to maintain stable ventilation. The findings of the present study suggest that the neural activity of the cortex can be changed on the basis of sensory perception and afferents information may be gated into the cerebral cortex. Therefore, a sensory gated model (Fig 5-1) is proposed to explain the cortical processing of respiratory afferents.

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96 Automatic breathing is regulated by brainstem respiratory center, which acts as an intrinsic controller to generate the respiratory motor activity. This respiratory motor activity is modified by the feedback from the chemoreceptors and mechanoreceptors to meet the respiratory demands. Afferent information from chemoreceptors increases or decreases the motor output until the arterial CO2 and O2 have returned to their set-points. On the other hand, afferents from respiratory mechanoreceptors modulate the central command of respiratory muscles to generate the optimal breathing pattern (Poon et al., 1992). Thus, respiratory afferent information modulates brainstem respiratory function but is normally gated-out of higher brain centers resulting in automatic ventilation with no cognitive awareness of breathing. If there is an error signal from chemoreceptors and/or mechanoreceptors in responding the disruption of the automatic breathing, such as inspiratory occlusion, the RREP will be elicited and the respiratory information will be perceived. This suggests that respiratory perception is normally gated-out in regular breathing and inspiratory occlusion may change the cortical neuronal activity resulting in gating-in of respiratory information to cognitive awareness. Thus, the automatic regulation of breathing can be overridden by a gate which modulates the conscious perception of respiration. This conscious awareness of breathing consists of the modulation in breathing pattern which involves the changes in arousal levels, emotions and cognitive activities (Ley, 1999). Thus, the gating modulation may also correlate to the association cortex (attention, thoughts and experiences) and limbic system (emotion). In the present study, the P1 peak amplitude of the RREP is not present with the increased background resistance because the applied resistive load is below the detection

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97 threshold. The fact that the RREP is not present with increased background resistance suggests that the respiratory resistive load information was gated-out by changing the background ventilatory state, such as increased background resistance. Although the interaction between the conscious and the automatic control of breathing is uncertain, a mismatch between respiratory afferent feedback on ventilatory status and the ventilatory demand will result in a cortical response and conscious awareness of breathing. Thus, a subcortical threshold must exist to gate-in or gate-out the respiratory afferent input and motor drive. However, neural indicators and mechanisms of the respiratory gating are unknown. Future study may explore the hypothetical construct of respiratory gating and the possible mechanisms that subserve the respiratory sensory gating system.

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98 Brainstem Respiratory Center GATE MOTOR CORTEX LIMBIC SYSTEM Emotions ASSOCIATION CORTEX Attention, Thoughts & Experience Respiratory Sensation and Perception Sensory Cortex Brainstem Sensory Relay Nuclei Spinal Cord Chemoreceptor Lung/Airway Afferents Airway Afferents Muscle Afferents Metabolic Load Volume Driving Pressure Tension Pump Motor Output Figure 5-1. Proposed respiratory gating model.

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CHAPTER 6 CONCLUSION The scalp distribution of RREP was similar to the mechanical stimulation elicited SEP. The RREP had features that were unique. The RREP P1 and N1 peaks corresponded to analogous peaks of the SEP and were recorded in the same location. However, the SEP P1 peak was unilateral while the RREP was bilateral. Although the recording techniques used in the current studies have limitations in dipole localization, the results of this study demonstrated the presence of the P1 peak of the RREP and SEP had the greatest amplitude in the electrodes overlying the somatosensory cortex which means these electrodes were in closest proximity to the dipole generator. These results are consistent with the P1 peak dipole located in the somatosensory cortex. Thus, the cortical distribution, neural mechanism and afferent pathways for cortical processing of respiratory mechanical load activation of the somatosensory cortex are supported by these RREP results. The inspiratory occlusion may have activated mechanoreceptors located in the respiratory system which may include the mouth, larynx, pharynx, lung, and respiratory pump muscles. Some or all of these mechanoreceptors may be mediating the activation of cortical neurons in the somatosensory region of the cerebral cortex. The P1 and N1 peaks of the RREP elicited by inspiratory occlusion were analogous to the corresponding peaks of the SEP elicited either by air puff or cutaneous mechanical stimulation. A similar cortical activation sequence was observed between the RREP and SEP except the Nf peak was only present in the frontal regions of the RREP. Cortical neurons activated by mechanical loads elicit a RREP similar to the SEP in the 99

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100 somatosensory system suggesting that these mechanoreceptor-activated neurons are in the somatosensory cortex and the information is mediated through the somatosensory pathway. Finally, the presence and loss of the RREP peaks without and then with increased background load, respectively, support the correlation of the RREP with the detection threshold for inspiratory resistive loads. The P1 peak was only present for those loads that exceeded the detection threshold. This was true even when background resistance was applied to change the detection threshold. The similarity of the latencies and amplitudes also suggested that perception of inspiratory loads is associated with the neural activation of the cortex and evoked potential is an objective measurement for perceptual information processing. In summary, the results from this dissertation suggested that inspiratory loads recruit multiple populations of respiratory mechanoreceptors. These afferents activate the somatosensory cortex. Cortical processing of respiratory loaded stimuli is a gated process with a threshold for cortical activation. Changing the respiratory status, such as the background resistance, changes the gate threshold requiring a greater stimulus to activate the cortex and elicit load detection.

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BIOGRAPHICAL SKETCH Yang-Ling Chou was born in I-Lan, Taiwan. She received her Bachelor of Physical Therapy degree from Kaohsiung Medical University in 1996. She joined the respiratory neurophysiological lab in the Department of Physiological Sciences of the University of Florida in January 2002 to pursue a Doctor of Philosophy degree, and since then she has worked as a research assistant in the Department of Physiological Sciences. Her research interests include respiratory sensation, control of breathing, and neurophysiology of respiration. 112