tracheal occlusion evoked respiratory load ...special thanks go to my parents, mr. chung-shun tsai...

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TRACHEAL OCCLUSION EVOKED RESPIRATORY LOAD COMPENSATION AND INHIBITORY NEUROTRANSMITTER EXPRESSION IN THE NUCLEUS OF SOLITARY TRACT IN ANIMALS AND THE EFFECT OF EMOTION ON THE

PERCEPTION OF RESPIRATORY LOAD IN HUMANS

By

HSIU-WEN TSAI

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

2013

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© 2013 Hsiu-Wen Tsai

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To my parents and my husband

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ACKNOWLEDGMENTS

This dissertation is a result of the hard work and support of many people. I was

fortune to be advised and mentored by Dr. Paul Davenport, my dissertation committee

chair. He has been an enthusiastic educator and wise leader throughout my PhD career.

I admire his intellect, dedication and focus in teaching and mentoring. I was privileged to

have a dissertation committee matching my research interests. My sincere gratitude is

given to my supervisory committee members: Dr. Donald Bolser, Dr. David Fuller, Dr.

Linda Hayward, Dr. Roger Reep and Dr. Christine Sapienza for their helpful suggestions

and advices that made the dissertation more meaningful and complete.

I am grateful to have the opportunity to work with all the members in our respiratory

lab: Dr. Kate Pate, Dr. Karen Wheeler-Hegland, Dr. Barbara Smith, Mark Hotchkiss,

Poonam Jaiswal, Sherry Adams and Jillian Condrey for their assistant throughout

graduate school. I thank Dr. Andreas von Leupoldt for his helpful suggestions and

insights into psychology. I am grateful to Dr. Pei-Ying Sarah Chan for her help, support

and friendship. I also thank Dr. Kun-Ze Lee for his help and encouragement. In addition,

I would like to thank Dr. Vipa Bernhardt and Dr. Ana Bassit for their help and support

throughout my Ph.D career.

Special thanks go to my parents, Mr. Chung-Shun Tsai and Mrs. Guei-Hua Tai for

their love, encourage, and tremendous sacrifices. No words can ever describe my

gratitude for all they have done. I thank my husband, Pang-Wei Liu. I could not come so

far without his continuous support and abiding love. Finally, I would like to thank Mrs.

Cherith Davenport for her care and support in my life in United State.

Last but not least, I would like to thank all my friends in Taiwan and at UF for

supporting me throughout these years.

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TABLE OF CONTENTS

page

ACKNOWLEDGMENTS .................................................................................................. 4

TABLE OF CONTENTS .................................................................................................. 5

LIST OF TABLES ............................................................................................................ 8

LIST OF FIGURES .......................................................................................................... 9

ABSTRACT ................................................................................................................... 11

CHAPTER

1 INTRODUCTION .................................................................................................... 13

Respiratory Load Compensation ............................................................................ 13

Central Control of Breathing ................................................................................... 15

Neurotransmitters in the Control of Breathing ......................................................... 20

The Nucleus of the Solitary Tract ............................................................................ 23

Specific Aims .......................................................................................................... 25

Specific Aim 1. Identification of Inhibitory Neurotransmitters in ITTO-Activated Neurons in the NTS of Anesthetized Rats. .................................... 25

Specific Aim 2. Identification of Inhibitory Neurotransmitters in NTS Neurons Activated by ETTO in Anesthetized Rats with Vagi Intact and Vagotomized. ................................................................................................ 26

Specific Aim 3. Identification of the Load Compensation Responses and the Inhibitory Neurotransmitters in the ITTO-Activated Neurons in the NTS of Conscious Rats. ............................................................................................ 27

Specific Aim 4. Identification of the Effect of Different Emotional States on the Perception of Graded Respiratory Resistive Loads in Conscious Humans. ........................................................................................................ 28

2 THE EFFECT OF TRACHEAL OCCLUSIONS ON RESPIRATORY LOAD COMPENSATION AND CHANGES IN NEURONS CONTAINING INHIBITORY NEUROTRANSMITTER IN THE NUCLEUS OF THE SOLITARY TRACT IN ANESTHETIZED RATS .......................................................................................... 30

Background ............................................................................................................. 30

Materials and Methods ............................................................................................ 32

Animals ............................................................................................................. 32

Surgical Process .............................................................................................. 32

Experimental Protocol ...................................................................................... 34

Immunofluorescence Double-Staining Protocol ................................................ 34

Data Analysis ................................................................................................... 35

Breathing pattern ....................................................................................... 35

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Immunofluorescence double-staining ........................................................ 36

Statistical analysis ...................................................................................... 37

Results .................................................................................................................... 37

Breathing Pattern ............................................................................................. 37

Immunofluorescence Double-Staining .............................................................. 38

Discussion .............................................................................................................. 39



3 THE EFFECT OF EXTERNAL TRACHEAL OCCLUSION ON THE LOAD COMPENSATION AND THE CHANGES IN INHIBITORY NEUROTRANSMITTER IN BRAINSTEM AFTER VAGOTOMY IN ANESTHETIZED RATS .......................................................................................... 52

Background ............................................................................................................. 52


Animals ............................................................................................................. 53

Surgical Procedure ........................................................................................... 53



Data Analysis ................................................................................................... 56

Statistical Analysis ............................................................................................ 56

Results .................................................................................................................... 56



Discussion .............................................................................................................. 59



4 THE EFFECT OF TRACHEAL OCCLUSION ON RESPIRATORY LOAD COMPENSATION AND CHANGES IN NEURONS CONTAINING INHIBITORY NEUROTRANSMITTER IN THE NUCLEUS OF THE SOLITARY TRACT IN CONSCIOUS RATS................................................................................................ 76

Background ............................................................................................................. 76


Animals ............................................................................................................. 79

Surgical Procedure ........................................................................................... 79



Data Analysis ................................................................................................... 81


Results .................................................................................................................... 81


lmmunofluorescence Double-Staining .............................................................. 81

Discussion .............................................................................................................. 82


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5 THE IMPACT OF EMOTION ON THE PERCEPTION OF GRADED MAGNITUDES OF RESPIRATORY RESISTIVE LOADS ....................................... 95

Background ............................................................................................................. 95


Participants ....................................................................................................... 97

Affective Picture Series .................................................................................... 97

Inspiratory Resistive Loads .............................................................................. 97

Measurement of Respiration ............................................................................ 98

Measurement of Emotional Responses ............................................................ 98

Procedure ......................................................................................................... 98


Results .................................................................................................................... 99

Discussion ............................................................................................................ 101

6 SUMMARIES AND CONCLUSIONS .................................................................... 110

Summary of Study Findings .................................................................................. 110

Study #1 Summary ......................................................................................... 110

Study #2 Summary ......................................................................................... 110

Study #3 Summary ......................................................................................... 111

Study #4 Summary ......................................................................................... 111

Respiratory Load Compensation in Anesthetized and Conscious Animals ........... 112

Effect of Emotion on Respiratory Perception ........................................................ 114

Activation of Inhibitory Glycinergic Neurons in the NTS ........................................ 114

Significance and Application of the Study ............................................................. 115

Direction for Future Studies .................................................................................. 117

Conclusions .......................................................................................................... 117

LIST OF REFERENCES ............................................................................................. 118

BIOGRAPHICAL SKETCH .......................................................................................... 129

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LIST OF TABLES

Table page 2-1 Comparisons of Ti, Te, Tt, Pes and EMGdia between different breaths of C,

O and R phases in anesthetized animals. .......................................................... 43

3-1 Comparisons of Ti, Te, Tt, Pes and EMGdia between different breaths of C, O and R phases in anesthetized animals with intact vagi. .................................. 62

3-2 Comparisons of Ti, Te, Tt, Pes and EMGdia between different breaths of C, O and R phases in vagotomized animals. .......................................................... 63

4-1 Comparisons of Ti, Te, Tt, Pes and EMGdia between different breaths of C, O and R phases in conscious animals. ............................................................... 86

5-1 Baseline characteristics of participants (Mean, SD) ......................................... 104

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LIST OF FIGURES

Figure page 1-1 Computational model of the brainstem respiratory network. ............................... 29

2-1 Effect of ITTO (shadowed area) on the respiratory load compensation .............. 44

2-2 ITTO elicited the load compensation responses in anesthetized animals. ......... 45

2-3 ITTO effect on EMGdia activity and Pes in anesthetized animals. ..................... 46

2-4 ITTO activated inhibitory glycinergic neurons in the cNTS in anesthetized animals. .............................................................................................................. 47

2-5 Immunofluorescence double-staining of c-Fos and GlyT2 in the cNTS in anesthetized animals. ......................................................................................... 48

2-6 ITTO activated inhibitory glycinergic neurons in the rNTS in anesthetized animals. .............................................................................................................. 49

2-7 Immunofluorescence double-staining of c-Fos and GlyT2 in the rNTS in anesthetized animals. ......................................................................................... 50

2-8 Immunofluorescence double-staining of c-Fos and GlyT2 in the combined rNTS and cNTS in anesthetized animals. ........................................................... 51

3-1 Effect of vagotomy on the respiratory load compensation responses. ................ 64

3-2 ETTO elicited load compensation responses in anesthetized animals with intact vagi. .......................................................................................................... 65

3-3 ETTO effect on EMGdia activity and Pes in anesthetized animals with intact vagi. .................................................................................................................... 66

3-4 Vagotomy load compensation responses with ETTO in anesthetized animals. .. 67

3-5 ETTO effect on Pes and EMGdia in vagotomized animals. ................................ 68

3-6 ETTO activated inhibitory glycinergic neurons in the cNTS in anesthetized animals with intact vagi. ...................................................................................... 69

3-7 The effect of vagotomy on the activation of inhibitory glycinergic neurons in the cNTS in anesthetized animals ...................................................................... 70

3-8 Immunofluorescence double-staining of c-Fos and GlyT2 in the cNTS in anesthetized animals with or without intact vagi. ................................................ 71

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3-9 ETTO activated inhibitory glycinergic neurons in the rNTS in anesthetized animals with intact vagi. ...................................................................................... 72

3-10 Effect of vagotomy on the activation of inhibitory glycinergic neurons in the rNTS in anesthetized animals. ............................................................................ 73

3-11 Immunofluorescence double-staining of c-Fos and GlyT2 in the rNTS in anesthetized animals with intact vagi and vagotomy. ......................................... 74

3-12 Immunofluorescence double-staining of c-Fos and GlyT2 in the combined rNTS and cNTS in anesthetized animals with intact vagi and vagotomy. ........... 75

4-1 Sustained ITTO behavior modulation of breathing pattern in conscious animals. .............................................................................................................. 87

4-2 Effect of sustained ITTO in conscious animals. .................................................. 88

4-3 Sustained ITTO effect on EMGdia in conscious animals. ................................... 89

4-4 The effect of ITTO on the activation of inhibitory glycinergic neurons in the cNTS in conscious animals. ............................................................................... 90

4-5 Immunofluorescence double-staining of c-Fos and GlyT2 in the cNTS in conscious animals. ............................................................................................. 91

4-6 ITTO activated inhibitory glycinergic neurons in the rNTS in conscious animals. .............................................................................................................. 92

4-7 Immunofluorescence double-staining of c-Fos and GlyT2 in the rNTS in conscious animals. ............................................................................................. 93

4-8 Immunofluorescence double-staining of c-Fos and GlyT2 in the combined rNTS and cNTS in conscious animals. ............................................................... 94

5-1 Schematic of experimental setup ...................................................................... 105

5-2 Mean SAM ratings of valence and arousal. ...................................................... 106

5-3 Peak mouth pressure Pmax and peak airflow at different levels of load resistance during pleasant, neutral and unpleasant picture series. .................. 107

5-4 The LogME–LogPmax relationship for the five resistive loads during pleasant, neutral and unpleasant picture series. .............................................. 108

5-5 Mean of slope of LogME-LogPmax for the pleasant, neutral and unpleasant picture series. ................................................................................................... 109

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Abstract of Dissertation Presented to the Graduate School of the University of Florida Partial Fulfillment of the

Requirements for the Degree of Doctor of Philosophy

TRACHEAL OCCLUSION EVOKED RESPIRATORY LOAD COMPENSATION AND INHIBITORY NEUROTRANSMITTER EXPRESSION IN THE NUCLEUS OF SOLITARY TRACT IN ANIMALS AND THE EFFECT OF EMOTION ON THE

PERCEPTION OF RESPIRATORY LOAD IN HUMANS

By

Hsiu-Wen Tsai

May 2013

Chair: Paul W. Davenport Major: Veterinary Medical Sciences

Animals are able to adapt to respiratory mechanical load by changing their

breathing pattern that is defined as respiratory load compensation. Load compensation

is mainly dominated by reciprocal inhibitory interconnections between brainstem

respiratory-related neurons in anesthetized animals but involves cortical and subcortical

processing in conscious animals. The nucleus of the solitary tract (NTS) is the principal

site that receives and integrates respiratory peripheral afferents for the neurogenesis of

load compensation. The central aim of this study is to investigate neurons that release

inhibitory neurotransmitters in the NTS that may be critically important for the

neurogenesis of load compensation in both anesthetized and conscious animals.

Furthermore, we investigated the effect of emotion on the graded respiratory load

perception in conscious humans.

The first study showed intrinsic, transient tracheal occlusions (ITTO) elicited load

compensation as evidenced by prolonged breath timing, e.g. inspiratory time (Ti),

expiratory time (Te), total breathing time (Ttot) and activated inhibitory glycinergic

neurons in the NTS of anesthetized animals. The second study demonstrated that

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external, transient tracheal occlusions (ETTO) also elicited load compensation with

prolongation of Ti, Te and Ttot, as well as activated glycinergic neurons in the NTS of

anesthetized animals. The load compensation responses were abolished by bilateral

cervical vagotomy and the activation of glycinergic neurons was suppressed in the

caudal NTS (cNTS) and rostral NTS (rNTS). The third study demonstrated in conscious

animals ITTO elicited behavioral control of load compensation with prolongation of only

Te and Ttot at the third occluded breath for each series of ITTO. Inhibitory glycinergic

neurons were only activated in the rNTS but not cNTS. The last study demonstrated

that emotional states modulate the behavioral load compensation by changing the

perception of graded magnitudes of respiratory resistive loads in conscious humans.

Negative emotional state increased the sense of respiratory effort for a single

presentation of low magnitude resistive load, but high magnitude loads were not

modulated by emotional states.

The results of these studies suggest that inhibitory glycinergic neurons in the

NTS play a significant role in the vagally-mediated neurogenesis of load compensation

in anesthetized animals that is greater than conscious animals. In conscious animals

and humans, cortical and subcortical processing is involved in the respiratory load

compensation responses.

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CHAPTER 1 INTRODUCTION

Respiratory Load Compensation

Breathing is a continuous movement of air in and out of the lung by inspiration and

expiration governed by a respiratory neural network. In the face of respiratory

mechanical or metabolic challenges, animals are able to compensate to the stimuli by

adjusting tidal volume (Vt) and/or breathing frequency (fb) to maintain appropriate

minute ventilation. Afferent inputs and motor outputs of the respiratory neural network

during eupneic breathing and respiratory load compensation have been extensively

studied in anesthetized animals. The neurotransmitters within the neurons of the

respiratory neural network responsible for the neurogeneis of breathing have been

studied. However, the neurons activated during respiratory load compensation

responses and the neurotransmitters within those neurons are unknown. It is also

unknown whether the changes in the respiratory neural network during load

compensation differ between anesthetized and conscious animals.

Respiratory load compensation was first characterized as the respiratory volume-

timing (Vt-T) relationship in anesthetized animals by Clark and von Euler (Clark and von

Euler, 1972). They demonstrated that Ti was inversely related to the Vt and Te was

dependent on Ti of the same breath. The Vt-T relationship was abolished by vagotomy

in anesthetized cats, indicating that the vagi are the principal respiratory afferents

transducing information related to the change of lung volume to the respiratory neural

network to elicit the Vt-T response. The Vt-T relationship has also been observed in

conscious humans, but only at a Vt at least two times of eupneic values (Clark and von

Euler, 1972). Subsequently, Zechman and colleagues (Zechman et al., 1976) used an

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extrinsic respiratory resistive loading model to study the respiratory reflex control

system in response to respiratory mechanical stimuli. They demonstrated that applying

a respiratory resistive load either to inspiration or expiration increased airway resistance

which in turn decreased inspired (Vi) or expired (Ve) air volume and resulted in a reflex

prolongation of Ti or Te of the loaded breath, respectively. Applying a single inspiratory

resistive load, the duration of the subsequent unloaded expiration was unchanged.

However, applying a single expiratory resistive load decreased inspiratory duration due

to an upward shift of functional residual capacity. The results imply that the Vt-T

relationship is controlled differently during inspiration and expiration.

Davenport et al. (Davenport et al., 1981a; Davenport et al., 1984) demonstrated that

slowly adapting pulmonary stretch receptors (PSRs), located in the smooth muscle of

the airways innervated by fast-conducting, myelinated afferent fibers in the vagus

nerves, respond to changes in either lung volume and smooth muscle tone and mediate

respiratory load compensation responses. PSRs are activated differently during

inspiration and expiration. During inspiration, lung inflation elevates PSR activity by

increasing the frequency of PSR discharge. During expiration, lung deflation activates

PSR by increasing the spike number (Davenport and Wozniak, 1986). Vagally-mediated

PSR afferents principally terminate on the second order interneurons in the intermediate

and caudal portions of the NTS (Kalia and Mesulam, 1980a) and project to the lateral

pons and ventral respiratory group (VRG) to modify the breathing pattern. Therefore,

the NTS is the principal structure to process peripheral respiratory afferents. It is

important to study the neurotransmitters released by interneurons in the NTS for further

understand the neurogenesis of respiratory load compensation responses.

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Central Control of Breathing

Autonomic respiration is a feedback control system composed of three elements:

brainstem central controller, effectors (respiratory muscles) and sensors (respiratory

afferents) (Bianchi et al., 1995). The interconnections between brainstem respiratory

neurons generate a rhythmic contraction of respiratory muscles to produce the

movements of inspiration and expiration which can be modulated by brainstem

respiratory neurons in response to respiratory afferent feedback.

Respiratory neurons in the brainstem can be characterized on the basis of their

discharge patterns, location and anatomic organization. On the basis of the location and

anatomic organization, respiratory brainstem neurons are mainly indentified as the

pontine respiratory group (PRG), dorsal respiratory group (DRG) and ventral respiratory

column (VRC) (Figure 1-1) (Rybak et al., 2007; Rybak et al., 2008; Smith et al., 2007).

In each respiratory group, respiratory neurons are further classified into various

subcategories according to the neuron’s discharge patterns, the time in a breathing

cycle at which they reach their maximum discharge frequency and the phase of

respiration during which they are activated (Bianchi et al., 1995). Early-I neurons exhibit

a peak discharge in early inspiration and decline gradually throughout the inspiration.

Post-I neurons rapidly reach peak discharge frequency with a gradually decrementing

discharge pattern in the early phase of expiration. I-Augmenting (I-Aug or ramp I)

neurons reach maximal discharge frequency in late inspiration with an augmenting

discharge pattern throughout the inspiratory phase and then gradually terminate their

activity in the early phase of expiration. E-Augmenting (E-Aug or aug-E) neurons begin

to fire in expiration, increase their discharge frequency and reach peak discharge during

the late expiratory phase. Late-I neurons only discharge in late inspiration and early

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expiration and are considered to be respiratory phase transition neurons. Pre-I neurons

discharge at the end of expiration and the beginning of inspiration and may be

responsible for terminating expiration and initiating the onset of the next inspiration. EI

and IE neurons are responsible for the transition between expiration and inspiration.

The PRG is located in the Kolliker-Fuse nucleus and parabrachial complex in the

rostral dorsolateral pons; however, several areas in the ventrolateral pons are important

for controlling the respiratory phase transition. Pontine inspiratory, expiratory and IE

neurons extensively interconnect with the VRC to control the Ti and Te and respiratory

phase transition. Rybak et al. speculated that inhibitory pump cells (p-cells) in the NTS

presynaptically inhibit all the excitatory outputs from the VRC to the pons during eupnea

(Rybak et al., 2008).

The DRG is mainly composed of inspiratory-related bulbospinal and propriobulbar

neurons. The distribution of projections from the DRG is variable in different species. In

cats, 50-90% of the inspiratory neurons (ramp-I, early-I) send projections to the phrenic

and external intercostal motorneurons in the spinal cord (Berger, 1977; Cohen et al.,

1974; Lipski et al., 1983); whereas in rats, less than 20% of DRG neurons project to the

motorneurons in spinal cord (de Castro et al., 1994).

The VRC is located in the vetrolateral medulla and contains several nuclei, including

the Bötzinger complex (BötC), the pre-Bötzinger complex (pre-BötC), the rostral (rVRG)

and caudal (cVRG) parts of the VRG. The BötC neurons are the principal source of

expiratory inhibition during eupneic breathing. Post-I and aug-E neurons in the BötC are

mutually inhibited and make widely distributed inhibitory interconnections with other

respiratory components in the pons and medulla (Ezure and Manabe, 1988; Ezure et

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al., 2003b; Jiang and Lipski, 1990; Merrill et al., 1983; Saito et al., 2002; Tian et al.,

1999). Post-I and aug-E neurons in the BötC inhibit pre-I and early-I neurons in the pre-

BötC to inhibit inspiratory outputs during expiration. Pre-I neurons in the pre-BötC are

less inhibited by the post-I neurons in the BötC during the late expiration to participate in

the transition from expiration to inspiration. Pre-I neurons in the pre-BötC have intrinsic

persistent sodium current dependent (INap) rhythmogenic properties that are inactivated

by tonic excitatory inputs and phasic inhibition from post-I and aug-E neurons in the

BötC during normal breathing but operate phasically to generate intrinsic rhythmic

inspiratory activity under certain conditions such as hypoxia and hypercapnia (Rybak et

al., 2007; St-John et al., 2009, Smith et al., 2007).

Pre-I in the pre-BötC neurons activate ramp-I neurons in the rVRG and hypoglossal

motor neurons to initiate inspiration (Feldman and Del Negro, 2006; Koizumi and Smith,

2008; Rekling and Feldman, 1998; Smith et al., 2000; Smith et al., 1991). In addition,

early-I and late-I neurons in the pre-BötC provide inhibitory projections to other

components in the network to coordinate breathing pattern (Ezure, 1990; Sun et al.,

1998). The VRG contains mainly bilateral clusters of bulbospinal respiratory neurons

and is subdivided into rVRG and cVRG. Ramp-I neurons are predominantly present in

the rVRG and project to spinal phrenic and intercostal motorneurons to shape the

inspiratory motor outputs (Bianchi et al., 1995). The excitatory expiratory premotor

neurons (E-Aug) in the cVRG send projections to spinal thoracic and lumbar

motorneurons to generate the expiratory motor outputs (Ezure, 1990).

The respiratory control system is regulated by feedbacks from chemoreceptors and

mechanoreceptors. Central chemoreceptors in the retrotrapezoid nucleus (RTN) and

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peripheral chemoreceptors in the aortic and carotid bodies sense the changes in the

concentration of oxygen and carbon dioxide in the cerebrospinal fluid and blood.

Mechanoreceptors in the lungs and airways, including PSRs, rapidly-adapting receptors

(RARs) and C-fibers, monitor the changes in the airflow and the volume of air in the

lungs. Mechanoreceptors in the respiratory muscles such as muscle spindles, Golgi

tendon organs and joint receptors sense the changes in the tension and length of

respiratory muscles.

Respiratory load compensation responses are primarily related to vagal

mechanoreceptors. PSRs, RARs and bronchopulmonary C-fibers course through the

vagus nerves to connect with the respiratory central control neural network (Kubin et al.,

2006). PSRs in the smooth muscles of the airways are activated by lung inflation and

terminate on second order interneurons (pump cells: p-cells; inspiratory-β cells: Iβ) in

the ipisilateral intermediate, interstitial, ventral, and ventrolateral subnuclei of the NTS

(Kubin et al., 2006) . RARs are located in the mucosa of the airways. They are activated

by rapid lung inflation, lung deflation, inhalation of noxious chemicals and mainly

terminate on second order interneurons in the commissural and intermediate subnuclei

of the NTS (Kubin et al., 2006) . Bronchopulmonary C-fibers are activated by external

irritants and terminate in the parvicellular nucleus, medial and commissural nucleus of

the NTS and the area postrema (Kubin et al., 2006). These respiratory sensory inputs

are mainly terminated and processed in different subnuclei of the NTS. The second

order NTS interneurons in turn project widely throughout the PRG and VRC to shape

and modulate inspiratory and expiratory motor outputs as well as to produce normal

breathing pattern (Ezure et al., 2002).

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Respiratory muscles are composed of laryngeal and pump skeletal muscles.

Respiratory laryngeal muscles in the upper airways regulate the airway resistance

during inspiration and expiration. The diaphragm, intercostals and abdominal muscles

are respiratory pump muscles. The diaphragm and external intercostals are the major

inspiratory muscles innervated by phrenic and intercostal motorneurons in the spinal

cord, respectively. The internal intercostals and abdominal muscles are the principal

expiratory muscles innervated by thoracic and lumbar motorneurons, respectively. The

movements of the respiratory laryngeal and pump muscles generate the inspiratory and

expiratory phases of breathing as a result of the coordination of respiratory neural

control network and integration of respiratory afferent feedbacks. In summary, the

synaptic interconnections within the brainstem central controller, respiratory muscles

and lung and airway receptors establish and regulate the rhythm and pattern of

breathing.

Breathing pattern is not only reflexively modified by brainstem feedback mechanism,

but can also be behaviorally altered. In conscious animals and humans, cortical and

subcortical structures are involved in the voluntary control of breathing. It has been

demonstrated that there is a respiratory gate system in the thalamus which determine

whether respiratory afferents will or will not reach the higher cortical processing areas

(Chan and Davenport, 2008; Davenport and Vovk, 2009). An appropriate respiratory

gate system is considered to be a protective mechanism for homeostasis that helps

animals to be aware and behaviorally adjust their breathing pattern in response to

respiratory challenges. Respiratory afferents that are gated into the cortical and

subcortical regions mainly project to the prefrontal cortex, limbic system and

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somatosensory cortex. Somatosensory areas integrate these signals and send

projections to the motor cortex. The motor cortex controls the respiratory muscles

through the corticospinal pathway to generate voluntary control of breathing (Davenport

and Vovk, 2009).

Neurotransmitters in the Control of Breathing

Neurons in the respiratory neural control network utilize neurotransmitters as

mediators for signal transmission to generate normal rhythmic breathing patterns. It is of

critical importance to identify the neurotransmitters which a neuron releases to define

the role it plays within the respiratory neural control network. Electrophysiological

techniques and immunohistochemistry staining have been used to identify the chemical

neuroanatomy of breathing in the brainstem. In electrophysiology, neurons are initially

characterized as excitatory or inhibitory by measuring the excitatory (EPSPs) or

inhibitory (IPSPs) post-synaptic potentials elicited from an identified neuron,

respectively. Immunohistochemistry staining is capable of identifying the morphology of

a neuron including its dendritic fields, axonal projections and the neurotransmitters it

secretes. A variety of studies combined these two methods to identify the chemical

neuroanatomy of breathing in the brainstem (Alheid and McCrimmon, 2008; Stornetta,

2008).

There are three major neurotransmitters utilized in the brainstem respiratory circuit:

glutamate, gamma-aminobutyric acid (GABA) and glycine. Glutamate is the major

excitatory neurotransmitter for the transmission of inspiratory drive in respiratory

premotor and motor neurons (Bonham, 1995; Haxhiu et al., 2005). Glutamate mainly

acts at non-N-methyl-D aspartate (non-NMDA) receptors, such as α-amino-3-hydroxy -

5-methylisoxazole-4-propionic acid (AMPA) and kainate within the network. Most of the

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cVRG bulbospinal neurons with a firing pattern that increased during expiration (aug-E)

were observed to be excitatory (Arita et al., 1987; Ballantyne and Richter, 1986). In the

NTS, bulbospinal inspiratory neurons appear to be excitatory (Cohen and Feldman,

1984; Cohen et al., 1974). In addition, peripheral chemoreceptors relayed to second

order interneurons in the commissural subnucleus of the NTS are mainly excitatory

(Finley and Katz, 1992; Otake et al., 1993).

Inhibitory neurotransmitters play a critical role in controlling neuronal excitability in the

respiratory neural network. GABA mainly acts on GABAA receptors and glycine binds to

glycine receptors that are chloride ion channels (Jentsch et al., 2002). Fast inhibitory

synaptic function of GABA and glycine is from the fast influx of chloride into the neuron

once their receptors are bound and activated. The negative charge inside the

membrane and positive charge outside the membrane of a neuron prevent it from

responding to further stimuli. Results from immunohistochemistry show that GABAergic

neurons are more densely distributed in the rostral brainstem and glycinergic neurons

are mainly located in the lower brainstem and spinal cord (Elekes et al., 1986; Lloyd et

al., 1983). It has been demonstrated that all types of medullary respiratory neurons

receive inhibitory inputs during their silent periods (Richter et al., 1992). Early-I neurons

receive IPSPs during the post inspiratory stage. I-Aug neurons receive glycine- and

GABA-mediated IPSPs at post inspiratory and late expiratory periods, respectively

(Richter et al., 1992). Aug-E neurons receive IPSPs during inspiratory phase and post-I

neurons during late expiration and early inspiration.

However, inspiratory neurons also receive IPSPs during inspiration and post-I

neurons receive IPSPs during late expiration (Ballantyne and Richter, 1984, 1986). It is

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apparent that GABA and glycine-mediated postsynaptic inhibitory inputs not only

prevent spiking of neurons during silent periods but also act in concert with excitatory

synaptic inputs during the active periods to help shape the pattern of the respiratory

motor outputs (Haji et al., 1990; Haji et al., 1992).

There is increasing evidence showing the importance of glycinergic inhibition in the

network of respiratory rhythm and pattern generation (Dutschmann and Paton, 2002a,

b; Ezure and Manabe, 1988; Iizuka, 1999; Saito et al., 2002; Schreihofer et al., 1999).

Reducing the concentration of chloride in the perfusate of an in situ arterially perfused

mature rat preparation abolishes the respiratory rhythm by a reduction of synaptic

inhibition (Hayashi and Lipski, 1992). Blockade of chloride-dependent synaptic inhibition

with glycine and GABAA receptor antagonists stops respiratory rhythmogenesis in

mature animals (Paton et al., 1994; Paton and Richter, 1995). Local application of

glycine by microinjection to the respiratory-related area in the brainstem decreases the

firing rate of bulbar respiratory neurons (Bianchi et al., 1995). Application of the glycine

antagonist strychnine depolarizes respiratory neurons in all phases of respiration

indicating that these neurons are tonically inhibited during breathing (Bianchi et al.,

1995). In the brainstem respiratory network, a variety of respiratory neurons secrete

glycine. Aug-E neurons in the BötC are glycinergic (Schreihofer et al., 1999). Post-I

neurons in the ventrolateral medulla secrete glycine (Ezure et al., 2003a). In addition,

the PSR activated second order interneurons in the ventrolateral interstitial subnucleus

of the NTS appear to be inhibitory and mainly secrete GABA (Takakura et al., 2007) but

also co-release glycine (26%) (Kubin et al., 2006). Therefore, in the respiratory control

23

system, inhibitory inputs seem to be more important than the excitatory inputs in the

generation of a coordinated breathing rhythm and pattern.

The Nucleus of the Solitary Tract

The NTS in the dorsomedial medulla oblongata is subdivided into several regions

based on cytoarchitectonics, including the commissual (SolC), dorsal (SolD), dorsal

lateral (SolDL), gelatinosus (SolG), interstitial (SolI), intermediate (SolIM), medial

(SolM), ventral (SolV) and ventolateral (SolVL) (Kalia and Mesulam, 1980a, b; Kalia and

Sullivan, 1982). The NTS is the principal site that receives and integrates peripheral

afferents from the respiratory, cardiovascular, gastrointestinal and gustatory systems.

The afferent axons are in the facial, glossopharyngeal and vagus nerves projecting into

the central nervous system for homeostasis and autonomic regulation. Peripheral

afferents from different visceral systems terminate in different subdivisions of the NTS.

Afferents from the respiratory, cardiovascular and gastrointestinal systems mainly

terminate in the caudal two thirds of the NTS and afferents from the gustatory system

terminate in the rostral one third of NTS (Kalia and Mesulam, 1980a, b).

The NTS is composed of a variety of respiratory-related heterogeneous neurons,

including inspiratory neurons (de Castro et al., 1994; Ezure et al., 1988; Matsumoto et

al., 1997), RAR relay neurons, (Ezure and Tanaka, 2000), SAR relay neurons (Bonham

and McCrimmon, 1990; de Castro et al., 1994; Ezure et al., 2002; Saether et al., 1987),

chemosensitive neurons (Chitravanshi and Sapru, 1995) and lung deflation sensitive

relay neurons (Ezure and Tanaka, 2000). Additionally, neurons with respiratory-related

discharging patterns have also been found in the NTS (Subramanian et al., 2007).

Early-I neurons with decrementing discharge pattern and late-I with augmenting

discharge pattern are mainly found in the SolVL rostral to the obex. Inspiratory neurons

24

are found in the SolVL rostral and caudal to the obex. The late-I with decrementing

discharge pattern and post-I neurons are found in the SolC caudal to the obex.

Expiratory neurons, I/E and E/I neurons are mainly found in the SolVL rostral to the

obex (Subramanian et al., 2007). These studies suggest that the NTS is the principal

site of the first synapse in afferent elicited respiratory reflexes and might play an

important role in respiratory pattern configuration.

Stimulation of vagal afferents and lung inflation are demonstrated to activate SARs in

the lungs and airways that terminate on the second order interneurons, p-cells, in the

NTS to control breathing pattern. PSR activated p-cells are either excitatory or inhibitory

that distribute and function differently. Excitatory p-cells project to the lateral pons, Post-

I in the BotC, I/E, late-I in the pre-BotC and ramo-I in the VRG to facilitate expiration and

terminate inspiration. Inhibitory p-cells project to the caudal SolC, lateral pons and early-

I in the pre-BötC and rVRG to inhibit inspiration. (Kubin et al., 2006; Rybak et al., 2007;

Rybak et al., 2008; Subramanian et al., 2007)

Based on results from immunohistochemistry, most peripheral afferents projecting to

the NTS utilize glutamate as an excitatory neurortansmitter (Sykes et al., 1997). The

inhibitory neurotransmitter, glycine, is frequently distributed in the SolI, SolDL and SolVL

subnuclei of the NTS(Tanaka et al., 2003). The other inhibitory neurotransmitter, GABA,

is often found in the area postrema, SolM, SolDM, SolI and SolVL subnuclei of the NTS

(Fong et al., 2005; Tanaka et al., 2003). In addition, it has been demonstrated that there

are more GABAergic neurons in the rostral brainstem, whereas glycinergic neurons are

more densely located in the caudal brainstem and spinal cord (Araki et al., 1988; Nicoll

et al., 1990; Elekes et al., 1986; Lloyd et al., 1983). This implies that the inhibitory

25

neurotransmitters might play a critical role in the NTS for the integration of respiratory

afferents for the modification of breathing patterns.

The purpose of this study is to examine the distribution of neurons that release

inhibitory neurotransmitter in the NTS in response to respiratory resistive loading in both

anesthetized and conscious animals.

Specific Aims

Specific Aim 1. Identification of Inhibitory Neurotransmitters in ITTO-Activated Neurons in the NTS of Anesthetized Rats.

Rationale: ITTO is an animal model that has been created in our laboratory for modeling

load compensation responses in patients with respiratory obstructive diseases. ITTO

has been demonstrated to elicit load compensation responses in anesthetized animals,

including prolongation of Ti, Te as well as Ttot (Pate and Davenport, 2012b). Results

from immnohistochemistry using the molecular maker c-Fos showed that neurons were

activated by ITTO in specific structures in the brainstem, especially in the NTS (Pate

and Davenport, 2012b). However, this method only shows that a neuron was activated

but it is not able to distinguish if the neuron uses excitatory or inhibitory

neurotransmitters. The NTS is the primary region that integrates peripheral respiratory

afferent inputs to the central nervous system. The second order interneurons in the NTS

receive inputs from vagal mechanoreceptors in the lungs and airways. The NTS second

order interneurons then project to the PRG and VRG to modify ventilation. Activation of

neurons in the NTS by ITTO suggests a reconfiguration of respiratory neural network to

generate load compensation responses. The Vt-T load compensation reflex response to

respiratory loads is characterized by an inhibition of inspiration and prolongation of

expiratory duration. The brainstem respiratory neural network is mainly constructed by

26

inhibitory reciprocal interconnections rather than excitatory synapses (Rybak et al.,

2007; Rybak et al., 2008; Smith et al., 2007). Therefore, we hypothesized that ITTO will

elicit a load compensation modulation of breathing pattern and ITTO-activated neurons

in the NTS will be inhibitory glycinergic neurons in anesthetized animals.

Hypothesis:

Acute ITTO-activated neurons in the NTS of anesthetized rats will be inhibitory glycinergic neurons.

Specific Aim 2. Identification of Inhibitory Neurotransmitters in NTS Neurons Activated by ETTO in Anesthetized Rats with Vagi Intact and Vagotomized.

Rationale: The most common strategy used to simulate patients with respiratory

obstructive diseases is to apply an external respiratory resistive load to breathing (Clark

and von Euler, 1972; Zechman et al., 1976). Vagal afferents transmit the information on

the external respiratory loads to p-cells in the NTS for the neurogenesis of load

compensation responses. Bilateral vagotomy eliminated the Vt-T relationship of load

compensation in anesthetized animals (Clark and von Euler, 1972; Webb et al., 1994).

Although the sensory pathway and motor responses have been studied extensively, the

brainstem neurons activated during respiratory load compensation and the

neurotransmitters within those neurons are still unknown. ITTO activated glycinergic

neurons in the NTS in the first study (Chapter 2) suggests that these NTS glycinergic

neurons play potential roles in the neurogenesis of load compensation. Therefore, we

will examine if consecutive external respiratory loads will activate glycinergic neurons in

the NTS and if bilateral vagotomy will block the expression of NTS glycinergic neurons.

Hypotheses:

ETTO will activate inhibitory glycinergic neurons in the NTS of anesthetized rats with vagi intact.

27

Vagotomy will eliminate ETTO-elicited load compensation responses.

Vagotomy will eliminate ETTO-activated inhibitory glycinergic neurons in the NTS of anesthetized rats.

Specific Aim 3. Identification of the Load Compensation Responses and the Inhibitory Neurotransmitters in the ITTO-Activated Neurons in the NTS of Conscious Rats.

Rationale: Load compensation in conscious animals is more complex compared with

unconscious animals as it involves more cortical and subcortical structures than the

purely brainstem-mediated reflex (Davenport and Vovk, 2009). Consciousness and

behavior play a critical role in the modulation of breathing in response to respiratory

challenges. Therefore, the neurogenesis and responses of load compensation is

expected to be different between conscious and anesthetized animals. It has been

demonstrated that ITTO (Chapter 2) and ETTO (Chapter 3) in anesthetized rats elicited

brainstem-mediated reflex load compensation responses as evidence by prolonged Ti,

Te and Ttot, as well as increased the activation of inhibitory glycinergic neurons in the

NTS. Therefore, the purpose of this third study is to investigate the ITTO-elicited load

compensation response and examine if the inhibitory glycinergic neurons in the NTS

play an important role in the neurogenesis of load compensation responses in

conscious rats.

Hypotheses:

The pattern of ITTO-elicited load compensation in conscious rats will be different from anesthetized rats.

The expression and distribution of ITTO-activated inhibitory glycinergic neurons in the NTS will be different between conscious and anesthetized rats.

28

Specific Aim 4. Identification of the Effect of Different Emotional States on the Perception of Graded Respiratory Resistive Loads in Conscious Humans.

Rationale: In conscious individuals, behavioral load compensation responses are

generated by the coordination of respiratory sensation, brainstem reflexes and motor

function (Ezure and Tanaka, 2004). Appropriate respiratory sensation is important to

consciously monitor the homeostasis of body allowing behavioral changes in breathing

patterns. Emotional states have been demonstrated to affect the respiratory sensation

and breathing pattern regardless of ventilatory changes. High negative affectivity (NA)

caused over-perception of respiratory discomfort in patient with COPD and asthma (De

Peuter et al., 2008; Vogele and von Leupoldt, 2008). In addition, experimentally

induced, short lasting negative states elevated reports of respiratory sensations in

patients and healthy individuals (Affleck et al., 2000; von Leupoldt et al., 2011; von

Leupoldt et al., 2006b; von Leupoldt et al., 2008). Although the impact of different

emotional states has been extensively studied, it is still unclear if emotional modulation

of respiratory perception is different with graded levels of respiratory restriction. In the

present study, affective pictures will be used to elicit different emotional states in healthy

participants. The effect of different emotional states on the perception of grated external

respiratory resistive loads will be investigated.

Hypothesis:

Pleasant and unpleasant emotional states will affect the perceived magnitude of respiratory resistive loads in conscious healthy participants.

.

29

Figure 1-1. Computational model of the brainstem respiratory network. Blue circles and

arrows represent inhibition. Red circles and arrows represent excitation. (Rybak et al., 2008, Smith et al., 2007, Molkov et al., 2013, Smith et al., 2013)

Pi Pe

Pump cells

Lungs

Diaphragm

NTSRaphéPONS

MEDULLA

BötC Pre-BötC rVRG cVRG

Tonic drive

Tonic drive

RTN

Tonic

drive

post-I

post-Ie

aug-E

pre-I/I

early-I (I)

ramp-I

early-I (2)

Bulbospinal

premotor E

PN

AbN

PSRs

I

IEi

IEe

E

30

CHAPTER 2 THE EFFECT OF TRACHEAL OCCLUSIONS ON RESPIRATORY LOAD

COMPENSATION AND CHANGES IN NEURONS CONTAINING INHIBITORY NEUROTRANSMITTER IN THE NUCLEUS OF THE SOLITARY TRACT IN

ANESTHETIZED RATS

Background

Reciprocal inhibitory connections between brainstem respiratory-related neurons

have been believed to play important roles in shaping the rhythm and pattern of

breathing. It has been demonstrated that all types of medullary respiratory neurons

receive inhibitory inputs during their silent periods (e.g. non-firing periods) (Richter et

al., 1992). Early-I neurons are inhibited during the post inspiratory stage. I-Aug neurons

receive glycine- and GABA-mediated IPSPs at post inspiratory and late expiratory

periods, respectively (Richter et al., 1992). E-Aug neurons during inspiratory phase and

post-I neurons during late expiration and early inspiration receive IPSPs inputs.

Additionally, cranial motor neurons and bulbospinal neurons with excitatory

neurotransmitters receive IPSPs during both active and quiescent periods (Bianchi et al.,

1995; Ezure, 1990). This indicates that inhibitory neurotransmitters not only prevent

spiking of neurons during silent periods but also act in concert with excitatory synaptic

inputs during active periods to help shape the pattern of respiratory motor output (Haji et

al., 1990; Haji et al., 1992). Taken together, the inhibitory neurotransmitters play

important roles in the brainstem respiratory neural network for the generation of normal

breathing rhythm and pattern.

The respiratory neural network also coordinates breathing with other respiratory-

related reflexes such as cough, swallow, sneeze and vomit by means of reconfiguration

of the respiratory neuronal pattern in the brainstem (Ambalavanar et al., 2004). Studies

have shown that electrically stimulated cough and swallow activate neurons in the SolI

31

and SolVL subnuclei of the NTS, lateral tegmental field of the reticular formation, the

area postrema and the nucleus ambiguus in anesthetized cats (Ambalavanar et al.,

2004). Respiratory resistive loads have activated the neurons in the NTS, nucleus

ambiguus and periaqueductal gray in anesthetized rats (Pate and Davenport, 2012b).

The NTS neurons that were activated in these studies imply they might play critical roles

for the coordination of breathing and respiratory-related reflexes.

The NTS in the dorsal medial medulla oblongata is the primary relay structure for the

integration of respiratory, cardiovascular, gastrointestinal and gustatory peripheral

afferents traveling in the facial, glossopharyngeal and vagus nerves into the central

neural brainstem network for the maintenance of homeostasis. The NTS contains a

variety of respiratory-related neurons including inspiratory neurons (de Castro et al.,

1994; Ezure et al., 1988; Matsumoto et al., 1997), RAR (Ezure and Tanaka, 2000) and

PSR relay neurons (Bonham and McCrimmon, 1990; de Castro et al., 1994; Ezure et

al., 2002; Saether et al., 1987), chemosensitive neurons (Chitravanshi and Sapru, 1995)

and deflation sensitive neurons (Ezure and Tanaka, 2000). These neurons receive and

process information from the afferents innervating the lungs and airways and make

connections with other interneurons in the NTS or project to other respiratory-related

structures throughout the brainstem for the generation or modification of a variety of

respiratory-related reflexes.

In the face of respiratory mechanical challenges, animals adapt by changing their

breathing patterns including changing tidal volume and/or breathing frequency. This

change in Vt-T breathing pattern is called the respiratory load compensation response

(Clark and von Euler, 1972; Zechman et al., 1976). Termination of inspiration and

32

expiration is determined by the inspired and expired air volume and transmural pressure

across the airways, respectively (Zechman et al., 1976). PSRs in the lungs and airways

sense the changes in the lung volume during respiratory loading and their afferents

terminate on the p-cells or other interneurons in the NTS. Some p-cells synapse with

other interneurons in the NTS and some project directly to respiratory-related neurons in

the PRG and VRG to change breathing pattern.

In a previous study, ITTO was demonstrated to elicit load compensation responses in

anesthetized animals that included prolongation of Ti, Te and Ttot, as well as

augmented diaphragm activity (Pate and Davenport, 2012b). Additionally, ITTO

activated the interneurons in the NTS. In the present study, we hypothesized that these

ITTO-activated interneurons are glycinergic neurons. We used immunofluorescence

double-staining of c-Fos, a biomarker of activated neurons, and GlyT2, a biomarker of

glycinergic neurons to test our hypothesis.

Materials and Methods

Animals

Experiments were performed on 12 male Sprague-Dawley rats (320-380 g). The

animals were housed in animal care facility at University of Florida. They were exposed

to a 12 hr light / 12 hr dark cycle and with free access to food and water. The

experimental protocol was reviewed and approved by the Institutional Animal Care and

Use Committee of the University of Florida.

Surgical Process

All animals were anesthetized with urethane (1.3 g/kg, ip). Adequate anesthetic

depth was verified by the absence of a withdrawal reflex to a rear paw pinch. Additional

urethane (20 mg/ml) was supplemented as necessary until the experiment was

33

terminated. Body temperature was monitored with a rectal temperature probe (Harvard

Apparatus; Holliston, MA) and maintained at 37-39°C using a heating pad. Animals

were spontaneously breathing room air throughout the experiment.

Animals were placed in a supine position and the right femoral artery was cannulated

using a saline-filled catheter (polyethylene-50 tubing) connected to a pressure

transducer to monitor blood pressure. A saline-filled tube (polyethylene-90 tubing) was

passed through the mouth into the esophagus and connected to a pressure transducer

to measure esophageal pressure (Pes). The analog outputs were amplified (Stoelting;

Wood Dale, IL), digitized at 5 kHz (Cambridge Electronics Designs 1401 computer

interface; Cambridge, UK), computer processed (Spike2, Cambridge Electronics

Design; Cambridge, UK) and stored for analysis. Pleural pressure changes were

inferred from relative changes in Pes.

Diaphragm electromyography (EMGdia) was recorded with bipolar Teflon-coated wire

electrodes. The distal ends of the wires were bared and bent to form a hook. The bared

tips of the electrodes were inserted into the costal diaphragm through a small incision in

the abdominal skin. The electrodes wires were connected to a high-impedance probe.

The signal was led into an amplifier (P511, Grass Instruments; Quincy, MA) and band-

pass filtered (30-3000 Hz). The analog output was digitized at 5 kHz and processed as

described above.

The trachea was exposed through a ventral incision and separated from surrounding

soft tissue. An inflatable cuff (Fine Science Tools; Foster, CA) was sutured around the

trachea, two cartilage rings caudal to the larynx. The cuff was connected to a saline-

filled syringe via a rubber tube. An appropriate pressure applied with the syringe inflated

34

the cuff bladder to compress the trachea causing complete closing of the airway.

Removal of the pressure restored the trachea back to its original condition with no

interference to breathing.

Experimental Protocol

The Sprague-Dawley rats were randomly divided into experimental (n=6) and sham

(n=6) groups. The experimental group underwent surgical preparation, 90 min post-

surgical period, 10 min obstructed breathing and then 90 min post-occlusion breathing.

The occlusion was applied for 2-3 seconds separated by at least 10-15 unobstructed

breaths. Pes and EMGdia were monitored throughout the experiment to confirm the

onset and removal of occlusions. The sham group underwent surgical preparation and

180 min post-surgical period but did not receive obstructed breathing.

Animals were euthanized and perfused with saline and 4% paraformaldehyde. Brains

were harvested and fixed in 4% paraformaldehyde for 24 h and then transferred into a

solution of 30% sucrose in PBS. The fixed brains were frozen and sectioned coronally

into 40 μm thick sections with a cryostat (HM101, Carl Zeiss; Thornwood, NY) for

immunofluorescence double-staining.

Immunofluorescence Double-Staining Protocol

For each brain, every fourth section of tissue was used for immunofluorescence

double-staining for c-Fos and glycine transporter 2 (GlyT2). Free-floating sections were

blocked in 5% normal donkey serum in PBS + Triton X-100 (PBS-T) for 1hr, and then

incubated in a mixed solution of guinea pig anti-GlyT2 (1:1000 dilution, Millipore;

Billerica, MA ) and rabbit anti-c-Fos primary antibodies (1:200 dilution, Santa Cruz

Biotechnology; Santa Cruz, CA) diluted in DAKO antibody diluents ( DAKO; Carpinteria,

CA) for 36 hrs. The following day the tissue was washed three times with PBS-T, and

35

then incubated in secondary antibodies (1:200 dilution for Cy2-condugated donkey anti-

guinea pig IgG and 1:100 dilution for Cy3-condugated donkey anti-rabbit IgG; Jackson

ImmunoResearch; West Grove, PA) for 2 hrs. The tissues were washed with PBS-T

three times and then mounted on glass slides. Sides were air-dried and cover-slipped

with anti-fading medium (DAKO; Carpinteria, CA). Negative controls were performed in

the absence of the primary antibody and the results showed no c-Fos or GlyT2 positive

staining.

Data Analysis

Breathing pattern

Spike 2 software (Cambridge Electronics Design; Cambridge, UK) was used for the

analysis of breathing pattern. The EMGdia signals were rectified and integrated with a

time constant of 50 ms (Figure 2-1). The Ti, Te, Ttot and EMGdia amplitude for each

breath were measured from the integrated EMGdia signals. Ti was measured from the

onset to the peak of EMGdia activity (Figure 2-1). Te was measured from the peak of

EMGdia activity to the following onset of EMGdia activity (Figure 2-1). Ttot was the sum

of Ti and Te. The EMGdia amplitude was measured from baseline to peak. For the

experimental group, these respiratory parameters were analyzed for the three

unobstructed control breaths (C: C3, C2, C1) prior to occlusions, the first three occluded

breaths (O: O1, O2, O3) and the following three unobstructed recovery breaths (R: R1,

R2, R3). For the sham group, these respiratory parameters were measured at matched

time periods. Pes was measured as the difference from the baseline to the peak of Pes.

Ti, Te, Ttot, EMGdia amplitude and Pes of occluded and recovery breath were

averaged and then normalized by using mean values of the control breaths for each rat

in experimental group.

36

Immunofluorescence double-staining

c-Fos is a transcription factor located in the nucleus of a neuron. GlyT2 is a

membrane protein which is a specific biomarker for glycinergic neurons. A standard

fluorescence microscope with appropriate filters was used to visualize and image

positive fluorescence staining in these brain tissue slides. The positive c-Fos-like

immunoreactivity was defined as red stain in the nucleus of the cell. A positive

immunofluorescence double-staining of c-Fos and GlyT2 was characterized c-Fos in the

nucleus (red) and GlyT2 positive staining (green) in the cytoplasm of the same neuron.

Brain regions were defined by the Rat Stereotaxic Atlas (Paxinos and Watson, 1998).

Twenty brain slices were collected in the rNTS defined by the coordinates Bregma, -

12.5 to -13.3 mm and cNTS defined by the coordinates Bregma, -13.3 to -14.1 mm.

Three or four levels of each region (rNTS or cNTS) in each brain were used for the

immunofluorescence double-staining of c-Fos and GlyT2. Immunoreactive cells at three

or four levels of each region of each animal were counted manually in bilateral rNTS

and cNTS by an individual blinded to the groups. There were no statistically significant

differences in the numbers of positive c-Fos neuron and co-labeled c-Fos and GlyT2

neurons on either side of these regions so the cell counts were averaged at each level.

Cell counts from the three or four slices of the same region were summed and then

multiplied by 3/20 or 4/20 (depend on how many levels were used for

immunofluorescence double-staining of c-Fos and GlyT2) to generate an estimate of the

total cell counts in the entire rNTS or cNTS. The total cell counts in the entire rNTS and

cNTS of each animal were summed to generate the cell counts in the combined rNTS

and cNTS. The value of co-labeled c-Fos and GlyT2 divided by the positive c-Fos cell

37

number represents the percentage of c-Fos positive cells co-labeled with GlyT2 in each

region of each animal.

Statistical analysis

All respiratory parameters are represented as mean ± SE. One-way repeated

measures analysis of variance (RMAVOVA) with breath as a factor (control, occluded

and recovery breaths) was used for the analysis of normalized Ti, Te, Ttot, EMGdia and

Pes which were followed by post-hoc paired t-tests. One-way ANOVA was performed to

compare the immunoreactive cell numbers in each region between experimental and

sham groups. A Greenhouse-Geisser correction was applied in case of violated

sphericity assumptions with reported significance levels referring to corrected degrees

of freedom. The significance criterion for all analyses was set at p < 0.05.

Results

Breathing Pattern

Comparisons between C (C1-C3), O (O1-O3), and R (R1-R3) breaths in experimental

animals revealed Ti, Te, Ttot, and Pes were significantly different between C and O

phases, and between O and R phases, but not between C and R phases (df = 8, F =

4.022, ε= 0.356, p < 0.05 for Ti; df = 8, F = 20.029, p < 0.001 for Te; df = 8, F = 19.850,

p < 0.001 for Ttot; df =8, chi-square = 20.8, p < 0 .01 for Pes). In detail, tracheal

occlusions resulted in a significant prolongation in Ti (Table 2-1, Figure 2-2A) and Te

(Table 2-1, Figure 2-2B) during O phase compared with C and R breath phases.

Prolongation of Ti and Te contributed to a significant increase in Ttot during tracheal

occlusions compared to C and R phases (Table 2-1, Figure 2-2C). Pes was more

negative during O phase compared to C and R phases, and returned to baseline

38

immediately after termination of occlusions, (df = 8, F= 26.883, p < 0.001, ε = 0.36)

(Table 2-1, Figure 2-3A). There were no significant differences in EMGdia between the

three phases in the experimental animals (Table 2-1, Figure 2-3B). There were no

significant differences in Ti, Te, Ttot, EMGdia and Pes at matched time points in the

sham animals.

Immunofluorescence Double-Staining

In the cNTS, there were no significant differences in the number of c-Fos and co-

labeled c-Fos and GlyT2 cells between experimental and sham groups. There were

significant differences in the percentage of c-Fos positive cells co-labeled with GlyT2

between experimental and sham groups (df = 1, F = 25.502, p < 0.01). The percentage

of c-Fos positive cells co-labeled with GlyT2 in the experimental group was significantly

greater than the sham group (t(9)= 5.050, p < 0.001) (Figure 2-4, Figure 2-5).

In the rNTS, there was an increasing trend in the number of c-Fos cells in the

experimental group compared with the sham group, (df = 1, F = 3.862, p = 0.097).

There were significant differences in the number of co-labeled c-Fos and GlyT2 cells

and the percentage of c-Fos positive cells co-labeled with GlyT2 (df = 1, F = 13.059, p <

0.01 and df =1, F = 35.195, p < 0.001, respectively). The number of co-labeled c-Fos

and GlyT2 cells in the experimental group was significantly greater than the sham group

(t(8)= 3,614 p < 0.01). The percentage of c-Fos positive cells co-labeled with GlyT2 was

higher in the experimental group than the sham group (t(8) = 5.933, p < 0.001) (Figure

2-6, Figure 2-7).

In the combined rNTS and cNTS, the number of c-Fos cells and co-labeled c-Fos and

GlyT2 cells showed no significant differences between experimental and sham groups.

39

However, there were significant differences in the percentage of c-Fos positive cells co-

labeled with GlyT2 between experimental and sham groups (df =1, F = 34.285, p <

0.001). The percentage of c-Fos positive cells co-labeled with GlyT2 was higher in the

experimental group than the sham group (t(9) = 6.970, p < 0.001) (Figure 2-8).

Discussion

Breathing Pattern

In the present study, Ti, Te and Ttot were prolonged during ITTO and returned to

normal breathing pattern immediately after the removal of occlusions. The increased Ti

and Te contributed to a longer Ttot during ITTO resulting in a decreased breathing

frequency in accordance with a previous study (Pate and Davenport, 2012b). Pes was

more negative during occlusion compared with C and R breaths indicating that the

tracheal cuff was inflated sufficiently to collapse the lumen of the trachea resulting in an

increase in airway resistance. EMGdia amplitude was not affected by ITTO. Taken

together, these results demonstrated that ITTO successfully elicited respiratory load

compensation responses in anesthetized animals.

The critical aspect of these experiments was the use of a tracheal occluder to

intrinsically evoke respiratory load compensation. In previous studies, respiratory load

compensation was mostly elicited by breathing through an external respiratory

resistance manifold, re-breathing valves or by the application of brochoconstrictors

(Zechman et al., 1976). The former two methods do not increase airway resistance

intrinsically so do not simulate increased intrinsic airway resistance in patients with

respiratory obstructive diseases. Bronchoconstrictors reduce the radius of the airways

to increase the intrinsic resistance; however, also causes airway inflammation. In our

laboratory, a tracheal occluder was implanted around the trachea and inflating the cuff

40

increases airway resistance intrinsically by closing the lumen of trachea. This strategy

increases airway resistance and successfully elicits the load compensation responses

without causing airway inflammation.


To our knowledge, this is the first study that identifies glycinergic neurons in the NTS

involved in the respiratory load compensation reflex based on immunofluorescence

double-staining of c-Fos and GlyT2. The co-labeled c-Fos and GlyT2 cell counts and

the percentage of c-Fos positive cells co-labeled with GlyT2 in the rNTS were

significantly increased by ITTO demonstrating that inhibitory glycinergic neurons in the

rNTS were activated by ITTO. In the cNTS, there was no significant difference in the co-

labeled c-Fos and GlyT2 cell counts; however, the percentage of c-Fos positive cells co-

labeled with GlyT2 were higher in the experimental group than the sham group. This

suggests that the same number but different subset of cells were activated by ITTO,

thus recruited a higher percentage of glycinergic cells in total c-Fos postivie cells. In

other words, ITTO did not change the quantity but the constitution of these activated c-

Fos neurons in the cNTS.

Anatomical and physiological studies have reported that the vagus nerves terminate

in the medulla. The first order neurons of the vagus nerves are in the nodose and

jugular ganglia that enter the lateral medulla and transmit sensory information from the

lungs and airways to the second order neurons in the lateral and medial divisions of

NTS (Kalia and Mesulam, 1980a; Kalia and Sullivan, 1982; Kubin et al., 2006; Yu,

2005). In the present study, the distribution of ITTO-activated glycinergic neurons in the

NTS is in accordance with the topographic distribution of vagal afferent termination. It is

also consistent with the hypothesis that these cells are the vagally relayed second or

41

higher order interneurons involved in the genesis of load compensation responses. In

fact, a variety of respiratory receptors traveling through the vagus nerves terminate in

different subnuclei of the NTS including RARs, PSRs and c-fibers but only PSRs have

been demonstrated to participate in the load compensation responses (Davenport et al.,

1984; Kubin et al., 2006) . PSRs sense the changes in the lung volume and transmural

pressure across the airways and relay to the second order interneurons, p-cells or Iβ

cells, in the NTS during either normal or loaded breathing for the generation of

appropriate ventilation by changing the breathing pattern. In addition, PSRs-activated

second order interneurons synapse with higher order interneurons in the NTS and/or

brainstem respiratory neural network interneurons may involve the neurogenesis of load

compensation responses as well.

In this study, we used c-Fos as a maker for neuronal activation. Although c-Fos

immunostaining is a powerful technique for the study of functional pathways of different

systems in the neural network; only when neurons are activated do they express c-Fos,

while inhibited neurons do not. However, c-Fos immunostaining is not able to

differentiate between motor and sensory pathways since motor, sensory and

interneurons can express c-Fos when they are activated. The NTS is the major

structure receiving and processing sensory inputs and is composed of a variety of

interneurons. In the present study, we can exclude the possibility that these activated c-

Fos neurons are motor neurons, but we do not know if they are sensory or interneurons,

which could be investigated in future studies.

This study is significant since it is the first to examine the type of inhibitory neurons

activated in the complex brainstem neural network configured to produce load

42

compensation responses during respiratory mechanical challenges. This study

enhances our understanding of the potential role of inhibitory glycinergic interneurons in

the NTS and we propose that they may be responsible for the neurogenesis of the

respiratory load compensation Vt-T response.

43

Table 2-1. Comparisons of Ti, Te, Tt, Pes and EMGdia between different breaths of C, O and R phases in anesthetized animals. p- values for all pairwise combination of all conditions for Ti, Te, Tt, Pes and EMGdia. N/A represents not significant between groups.

Ti Te Tt Pes EMGdia

C1 vs. O1 N/A <.01 <.05 N/A N/A

C1 vs. O2 <.05 <.01 <.01 <.05 N/A

C1 vs. O3 N/A <.01 <.01 <.05 N/A

C1 vs. R1 N/A N/A N/A N/A N/A

C1 vs R2 N/A N/A N/A N/A N/A

C1 vs. R3 N/A N/A N/A N/A N/A

O1 vs. R1 N/A <.01 <.05 <.05 N/A

O1 vs. R2 N/A <.01 <.01 <.05 N/A

O1 vs. R3 N/A <.01 <.01 N/A N/A

O2 vs. R1 N/A <.01 <.01 <.05 N/A

O2 vs. R2 N/A <.01 <.01 <.05 N/A

O2 vs. R3 N/A <.01 <.01 <.05 N/A

O3 vs. R1 <.05 <.01 <.01 <.05 N/A

O3 vs. R2 N/A <.01 <.01 <.05 N/A

O3 vs. R3 <.05 <.01 <.01 <.05 N/A

44

Figure 2-1. Effect of ITTO (shadowed area) on the respiratory load compensation

Physiological results during control (C3-C1), occluded (O1-O3) and recovery (R1-R3) breaths. Determination of Ti and Te are shown in the trace of integrated EMGdia

45

A B

C

Figure 2-2. ITTO elicited the load compensation responses in anesthetized animals.

Normalized breath timing values during control (C3, C2, C1), occluded (O1, O2, O3) and recovery (R1, R2, R3) breaths. (A) The relationship between Ti and breath number. (B) The relationship between Te and breath number. (C) The relationship between Ttot and breath number. The * indicates a significant difference, p < 0.05; the ** indicates a significant difference, p < 0.01

Breath

C3 C2 C1 O1 O2 O3 R1 R2 R3

Insp

irato

ry T

ime (

Rela

tive U

nits

)

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

*

Breath

C3 C2 C1 O1 O2 O3 R1 R2 R3

Exp

irato

ry T

ime (

Rela

tive U

nits

)

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

** ** **Breath

C3 C2 C1 O1 O2 O3 R1 R2 R3

Tota

l Bre

ath

Tim

e (

Rela

tive U

nits

)

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

* ** **

46

A B

Figure 2-3. ITTO effect on EMGdia activity and Pes in anesthetized animals.

Normalized EMGdia (A) and Pes (B) during control (C3, C2, C1), occluded (O1, O2, O3) and recovery (R1, R2, R3) breaths. The * indicates a significant difference, p < 0.05

Breath

C3 C2 C1 O1 O2 O3 R1 R2 R3

EM

Gdia

(R

ela

tive U

nits

)

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

o

Breath

C3 C2 C1 O1 O2 O3 R1 R2 R3

Eso

phageal P

ress

ure

(R

ela

tive U

nits

)

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

* *

47

Figure 2-4. ITTO activated inhibitory glycinergic neurons in the cNTS in anesthetized

animals. Immunofluorescence double-staining of c-Fos (red) and GlyT2 (green) in the cNTS (Bregma -13.8 mm) in sham (B-G) and experimental groups (H-M). The dashed line in part A represents the area of rat brain atlas corresponding to the region in B-D and H-J. E-G and K-M represent the dashed area in B-D and H-J respectively. TS: Solitary Tract. Arrows represent immunoreactive cells. Photo courtesy of Hsiu-Wen Tsai

A

H I J

K L

TSTS TS

B C D

E F G

TS TSTS

c-Fos GlyT2 Merge

Sham

Exp

48

A B

C

Figure 2-5. Immunofluorescence double-staining of c-Fos and GlyT2 in the cNTS in anesthetized animals. (A) c-Fos labeled cell number (B) co-labeled c-Fos and GlyT2 cell number (C) the percentage of c-Fos positive cells co-labeled with GlyT2. The ** indicates a significant difference, p < 0.01

Groups

Sham Exp

c-F

os

labele

d c

ell

num

ber

0

200

400

600

800

1000

;

Groups

Sham Exp

c-F

os

and G

lyT

2 c

ola

bele

d c

ell

num

ber

0

200

400

600

800

1000

.

Groups

Sham Exp

c-F

os a

nd

Gly

T2

co

lab

ele

d c

ell

nu

mb

er

/ c-F

os la

be

led

ce

ll n

um

be

r

0

20

40

60

80

100

***

49

Figure 2-6. ITTO activated inhibitory glycinergic neurons in the rNTS in anesthetized animals. Immunofluorescence double-staining of c-Fos (red) and GlyT2 (green) in the rNTS (Bregma -13.2 mm) in sham (B-G) and experimental groups (H-M). The dashed line in part A represents the area of rat brain atlas corresponding to the region in B-D and H-J. E-G and K-M represent the dashed area in B-D and H-J respectively. TS: Solitary Tract. Arrows represent immunoreactive cells. Photo courtesy of Hsiu-Wen Tsai

TS TSTS

B C D

E F G

TS TSTS

H I J

K L M

c-Fos GlyT2 Merge

Sham

Exp

A

50

A B

C

Figure 2-7. Immunofluorescence double-staining of c-Fos and GlyT2 in the rNTS in anesthetized animals. (A) c-Fos labeled cell number (B) co-labeled c-Fos and GlyT2 cell number (C) the percentage of c-Fos positive cells co-labeled with GlyT2. The * indicates a significant difference, p < 0.05; the *** indicates a significant difference, p < 0.001

Groups

Sham Exp

c-F

os

labele

d c

ell

num

ber

0

200

400

600

800

1000

p = 0.097

Groups

Sham Exp

c-F

os

and G

lyT

2 c

ola

bele

d c

ell

num

ber

0

200

400

600

800

1000

**

Groups

Sham Exp

c-F

os a

nd

Gly

T2

co

lab

ele

d c

ell

nu

mb

er

/ c-F

os la

be

led

ce

ll n

um

be

r

0

20

40

60

80

100

***

51

A B

C

Figure 2-8 Immunofluorescence double-staining of c-Fos and GlyT2 in the combined rNTS and cNTS in anesthetized animals. (A) c-Fos labeled cell number (B) co-labeled c-Fos and GlyT2 cell number (C) the percentage of c-Fos positive cells co-labeled with GlyT2. The *** indicates a significant difference, p < .001

>

Groups

Sham Exp

c-F

os

labele

d c

ell

num

ber

0

200

400

600

800

1000

.

Groups

Sham Exp

c-F

os a

nd G

lyT

2 c

ola

bele

d c

ell

num

ber

0

200

400

600

800

1000

Groups

Sham Exp

c-F

os

and G

lyT

2 c

ola

bele

d c

ell

num

ber

/ c-

Fos

labele

d c

ell

num

ber

0

20

40

60

80

100

***

52

CHAPTER 3 THE EFFECT OF EXTERNAL TRACHEAL OCCLUSION ON THE LOAD

COMPENSATION AND THE CHANGES IN INHIBITORY NEUROTRANSMITTER IN BRAINSTEM AFTER VAGOTOMY IN ANESTHETIZED RATS

Background

Applying an external respiratory resistive load is one of the most common

strategies to study respiratory load compensation in animals to simulate the limitation of

airflow in patients with respiratory obstructive disease. Respiratory load compensation is

characterized by the Vt-T reflex in animals (Clark and von Euler, 1972; Zechman et al.,

1976). Inspiration and expiration were prolonged by inspiratory or expiratory loads,

respectively (Zechman et al., 1976). Augmented respiratory motor output is also

characterized as load compensation responses (Koehler and Bishop, 1979; Lopata et

al., 1983). Vagal afferents have been demonstrated to transmit the afferent activity

modulated by external respiratory loads to the NTS of brainstem for the neurogenesis of

respiratory load compensation response. Studies have shown that vagal afferents,

especially PSRs, respond to inspiratory and expiratory loads differently; bilateral

vagotomy eliminated the Vt-T response only during single inspiratory loading but not to

the same extent during single expiratory loading in anesthetized animals (Webb et al.,

1994, 1996). However, it is unknown if vagal afferents have the same effect on the

neurogenesis of load compensation response during sustained respiratory loading of

entire breaths.

Moreover, the sensory and motor pathways of respiratory load compensation

elicited by external respiratory loads have been studied extensively. The brainstem

neurons activated during the respiratory load compensation response and the

neurotransmitters within those neurons are still unknown. In the first study (Chapter 2), it

53

has been demonstrated that ITTO activated glycinergic neurons in the NTS. The results

suggest that inhibitory glycinergic neurons in the NTS may play a role in the

neurogenesis of load compensation in anesthetized animals. In the present study, we

determined if the load compensation response during sustained external respiratory

loads would be abolished by bilateral vagotomy. In addition, we investigated if the

glycinergic neurons in the NTS would be activated by sustained external respiratory

loads. Further, we hypothesized that bilateral cervical vagotomy would abolish load-

elicited activation of glycinergic neurons in the NTS.


Animals

Experiments were performed on 16 male Sprague-Dawley rats (320-380 g). The

animals were housed in the University of Florida animal care facility. They were

exposed to a 12 hr light/12 hr dark cycle and with free access to food and water. The



Surgical Procedure

The rats were randomly divided into four groups (A) vagi intact rats without external

and transient tracheal occlusions (ETTO), (B) vagi intact rats with ETTO, (C)

vagotomized rats without ETTO and (D) vagotomized rats with ETTO. Animals were

anesthetized with urethane (1.3 g/kg, ip). Adequate anesthetic depth was verified by the

absence of a withdrawal reflex to a rear paw pinch. Additional urethane (20 mg/ml) was

supplemented as necessary until the experiment was terminated. Body temperature

was monitored with a rectal temperature probe (Harvard Apparatus; Holliston, MA) and

54

maintained at 37-39°C using a heating pad. Animals were spontaneously breathing

room air throughout the experiment.

Animals were placed in a supine position and the right femoral artery was cannulated

using a saline-filled catheter (polyethylene-50 tubing) connected to a pressure

transducer to monitor blood pressure. A saline-filled tube (polyethylene-90 tubing) was

passed through the mouth into the esophagus and connected to a pressure transducer

to measure Pes. The analog outputs were amplified (Stoelting; Wood Dale, IL), digitized

at 5 kHz (Cambridge Electronics Designs 1401 computer interface; Cambridge, UK),

computer processed (Spike2, Cambridge Electronics Design; Cambridge, UK) and

stored for analysis. Pleural pressure changes were inferred from relative changes in

Pes.

Diaphragm electromyography (EMGdia) was recorded with bipolar Teflon-coated wire

electrodes. The distal ends of the wires were bared and bent to form a hook. The bared

tips of the electrodes were inserted into the costal diaphragm through a small incision in

the abdominal skin. The electrode wires were connected to a high-impedance probe.

The signal was fed into an amplifier (P511, Grass Instruments; Quincy, MA) and band-

pass filtered (30-3000 Hz). The analog output was digitized at 5 kHz and processed as

described above (Chapter 2).

The trachea was exposed through a ventral incision and separated from surrounding

soft tissue. Tracheotomy was performed in all animals. A tracheal cannula was inserted

into the tracheotomy. In addition, in group C and D, bilateral cervical vagus nerves were

separated from surrounding tissues and carotid artery and severed.

55


There were four groups in the present experiment. The group A was surgically

prepared but breathing was not obstructed. The group B was surgically prepared and

received 10 min of ETTO. The group C and group D were surgically prepared and

bilaterally cervical vagotomized. The group D received 10 min of ETTO but the tracheal

occlusion was not performed in group C.

The occlusion was applied for 2-3 seconds by blocking the outlet of the tracheal

cannula and then unblocking for at least 10 unobstructed breaths. Pes and EMGdia

were monitored throughout the experiment to confirm the onset and removal of

occlusions. The group B and D underwent surgical preparation and 90 min post-surgical

period but did not receive obstructed breathing.

Animals were maintained under anesthesia for 90 minutes following completion of the

experimental protocol. Following this 90 min period the animals were euthanized and

perfused. Brains were harvested and fixed in 4% paraformaldehyde for 24 h and then

transferred into a solution of 30% sucrose. The fixed tissue was coronally sectioned into

40 μm thick sections with a cryostat (HM101, Carl Zeiss) for immunofluorescence

double-staining analysis.


For each brain, every fourth section of tissue was used for immunofluorescence

double-staining for c-Fos and GlyT2. Free-floating sections were blocked in 5% normal

donkey serum in PBS + Triton X-100 (PBS-T) for 1h, and then incubated in a mixed

solution of guinea pig anti-GlyT2 (1:1000 dilution, Millipore; Billerica, MA ) and rabbit

anti-c-Fos primary antibodies (1:200 dilution, Santa Cruz Biotechnology; Santa Cruz,

CA) diluted in DAKO antibody diluents ( DAKO; Carpinteria, CA) for 36 hrs. The

56

following day the tissue was washed three times with PBS-T, and then incubated in

secondary antibodies (1:200 dilution for Alexa Fluor® 488-AffiniPure Donkey Anti-

Guinea Pig IgG and 1:100 dilution for Alexa Fluor 594-AffiniPure Donkey Anti-Rabbit

IgG; Jackson ImmunoResearch; West Grove, PA) for 2 hrs. Slices of tissue were

washed with PBS-T three times and then mounted on glass slides. Sides were air-dried

and cover-slipped with anti-fading medium (DAKO; Carpinteria, CA). Negative controls

were performed in the absence of the primary antibody and the results showed no c-Fos

or GlyT2 positive staining.

Data Analysis

The methods are the same as described in Chapter 2.

Statistical Analysis

All respiratory parameters are presented as mean ± SE. One-way repeated

measures analysis of variance (RMAVOVA) with breath as a factor (control, occluded

and recovery breaths) was used for the analysis of normalized Ti, Te, Ttot, EMGdia and

Pes which were followed by post-hoc paired t-tests. One-way ANOVA was performed to

compare the immunoreactive cell numbers in each region between these four groups. A

Greenhouse-Geisser correction was applied in case of violated sphericity assumptions

with reported significance levels referring to corrected degrees of freedom. The

significance criterion for all analyses was set at p < 0.05.

Results

Breathing Pattern

In group A and group C, there were no significant differences in Ti, Te, Ttot, EMGdia

and Pes between breaths during the experiment.

57

In group B, Ti, Te, Ttot and EMGdia showed significant differences between O, C and

R phases (df = 8, F = 9.419, p < 0.001 for Ti; df = 8, F = 16.740, p < 0.01, ε= 0.189 for

Te; df = 8, F = 16.702, p < 0.001 for Ttot). Ti was significantly increased during the O

phase compared with C and R phases (Table 3-1, Figure 3-1 top panel, Figure 3-2A).

Te was also prolonged during the O phase compared with C and R phases (Table 3-1,

Figure 3-1 top panel, Figure 3-2B). The prolongation of Ti and Te contributed to a longer

Ttot during the O phase compared with C and R phases (Table 3-1, Figure 3-1 top

panel, Figure 3-2C). Pes showed significant differences between the three phases (df =

8, F= 13.878, p < 0.01 for EMGdia; df = 8, F = 3.721, p < 0.001 for Pes). Pes was more

negative during occlusions than C and R phases. Pes returned to baseline during R

phase and was not significantly different from the C phase (Table 3-1, Figure 3-1 top

panel, Figure 3-3A).

In group D, there were no significant differences in Ti between the three phases

(Table 3-2, Figure 3-2 bottom panel, Figure 3-4A). Te and Ttot showed significant

differences between three phases (df = 8, chi-square = 27.0, p < .001 for Te; df = 8, chi-

square = 27.133, p < .001 for Ttot). Tracheal occlusions resulted in a significant

decrease in Te compared with C and R phases (Table 3-2, Figure 3-2 bottom panel,

Figure 3-4B). Shortening of Te contributed to a significant decrease in Ttot during O

phases compared to C and R phases (Table 3-2, Figure 3-2 bottom panel, Figure 3-4C).

Pes was more negative during O phase than C and R phases (df = 8, F= 356.523, p <

0.001) (Table 3-2, Figure 3-1 bottom panel, Figure 3-5A). There were no significant

differences in EMGia between C, O and R phases (Figure 3-5B).

58


Figure 3-6 and Figure 3-7 show the double-staining of c-Fos and GlyT2 in the cNTS

in the four groups. In the cNTS, no significant differences in the number of c-Fos cells

were observed among groups (Figure 3-8A). The number of co-labeled c-Fos and

GlyT2 cells showed significant differences among groups (df = 3, F = 8.86, p <

0.01).The number of co-labeled c-Fos and GlyT2 cells in group B was significant greater

than group A, C and D (group A vs. group B: t(6) = 3.961, p < 0.01; group B vs. group

C: t(6) = 5.173, p < 0.01; group B vs group D: t (6) = 2.689, p < 0.05) (Figure 3-8B).

There were significant differences in the percentage of c-Fos positive cells co-labeled

with GlyT2 among groups (df = 3, F = 15.075, p < 0.001). The percentage of c-Fos

positive cells co-labeled with GlyT2 in group B was significantly greater than group A, C

and D (group A vs. group B: t(6) = 6.641, p < 0.001; group B vs. group C: t(6) = 5.061, p

< 0.01; group B vs. group D: t (6) = 3.934, p < 0.01) (Figure 3-8C) but there were no

significant differences between groups A, C and D.

Figure 3-9 and Figure 3-10 show the double-staining of c-Fos and GlyT2 in the rNTS.

In the rNTS, there were no significant differences in the number of c-Fos cells among

the four groups (Figure 3-11A). There were significant differences in the number of co-

labeled c-Fos and GlyT2 cells between groups (df =3, F=4.886, p < 0.05) (Figure 3-11B).

The number of co-labeled c-Fos and GlyT2 cells in group B was significantly greater

than group A, C and trended higher than group D (group A vs. group B: t(6) = 2.613, p <

0.05; group B vs. group C: t(6) = 2.795, p < 0.05; group B vs. group D: t(6) = 1.867, p =

0.111). The percentage of c-Fos and GlyT2 co-labeled cells showed significant

differences among the four groups (df = 3, F = 21.226, p < 0.001) (Figure 3-11C). The

percentage of c-Fos positive cells co-labeled with GlyT2 was significantly greater in

59

groups B than group A, C and D (group A vs. group B: t(6) = 7.270, p < 0.001; group B

vs. group C: t(6) = 10.722 , p < 0.001; group B vs. group D: t(6) = 4.948, p = 0< 0.01).

For the combined rNTS and cNTS, the number of c-Fos cells showed no significant

difference among the four groups (Figure 3-12A). There were significant differences in

the number of co-labeled c-Fos and GlyT2 cells and the percentage of c-Fos positive

cells co-labeled with GlyT2 (df = 3, F = 10.668, p < 0.001 and df = 3, F = 23.586, p <

0.001, respectively) (Figure 3-12B and Figure 3-12C). The number of co-labeled c-Fos

and GlyT2 cells and the percentage of c-Fos positive cells co-labeled with GlyT2 in

group B were significantly greater than group A, C and D (group A vs. group B: t(6) =

4.396, p < 0.01; group B vs. group C: t(6) = 5.313, p < 0.001; group B vs. group D: t(6) =

3.020, p < 0.05). The percentage of c-Fos positive cells co-labeled with GlyT2 in group

B was significantly greater than group A, C and D (group A vs. group B: t(6) = 7.505, p <

0.001; group B vs. group C: t(6) = 8.067, p < 0.001; group B vs. group D: t(6) = 4.741, p

< 0.01).

Discussion

Breathing Pattern

In group B, prolongation of Ti, Te, Ttot, and increased amplitude of EMGdia during O

phase indicates the respiratory load compensation responses were elicited by external

respiratory resistive loads.

In vagotomized rats, Ti was not affected by tracheal occlusions and Te and Ttot

during O phase were even slightly shorter than during C and R phase in group D

indicating that respiratory load compensation responses were suppressed by bilateral

cervical vagotomy. During C and R phases, the breathing pattern in vagtomized animals

was slower than animals with intact vagi. This is a typical breathing pattern in

60

vagotomized animals due to the interruption of transmission of afferent activity from

PSRs during inspiration to terminate inspiratory effort; therefore the central brainstem is

mediating the control of breathing rhythm and pattern. In addition, the shorter Te and

Ttot during O1 compared to C and R breaths suggests other extravagal mechanisms,

such as afferents from the respiratory muscles or other non-vagal respiratory afferents

contribute to the modulation of breathing patterns (Campbell et al., 1964; Corda et al.,

1965; Lynne-Davies et al., 1971).


To our knowledge, this is the first study demonstrating that inhibitory glycinergic

neurons in the cNTS and rNTS were activated by external respiratory resistive loads.

These activated glycinergic neurons were concentrated in the SolM and SolV subnuclei

of the NTS. The results were consistent with the first study (Chapter 2) and show that

both ITTO and ETTO activated glycinergic neurons in the same subdivisions of the NTS.

These activated glycinergic neurons in the NTS might be considered as the second

order interneurons possibly- p-cells or p-cell related higher order interneurons. First, the

afferents of PSRs in the lungs and airways that are responsible for a variety of

respiratory reflexes, such as inspiratory termination, expiratory facilitation, enhancement

of inspiratory effort and bronchodilation (Kubin et al., 2006) mainly synapse with p-cells

in the SolIM, SolI, SolV and SolVL subnuclei of the NTS. Second, two-thirds of pump

cells in the NTS use inhibitory GABA and glycine as neurotransmitters (Ezure and

Tanaka, 2004). Application of excitatory amino acids on the SolM resulted in reflex

termination of inspiration and prolongation of expiration while blockade of excitatory

amino acids in this area reduced these changes (Bonham et al., 1993; Bonham and

McCrimmon, 1990). Finally, these glycinergic neurons are consistent with the role of

61

inhibitory p-cells in the brainstem respiratory network proposed by Rybak et al. (Rybak

et al., 2007; Rybak et al., 2008). They modeled these inhibitory p-cells to excite I-Dec

neurons in the BötC for the termination of inspiration and prolongation of expiration and

presynaptically inhibit inspiratory neurons in the pons for the control of breathing pattern.

Therefore, the potential role of the activated glycinergic neurons in the NTS might be

related to the neurogenesis of load compensation responses.

The percentage of c-Fos positive cells co-labeled with GlyT2 in the cNTS and rNTS

was abolished by bilateral cervical vagotomy. Kalia et al. have demonstrated that vagal

afferents enter the lateral medulla from Bregma -12.8 to -15.3 mm and terminate in

different subdivisions of the NTS from Bregma -11.8 to -18.3 mm (Kalia and Mesulam,

1980a). In the present study, the rNTS was defined as the region from Bregma -12.5 to

-13.3 mm and cNTS was from Bregma -13.3 to -14.1 mm, thus including the area of

entry and termination of vagal afferents. Therefore, the present results demonstrate that

bilateral cervical vagotomy could successfully block transmission of information from the

PSRs in the lungs and airways to glycinergic neurons in the NTS normally activated by

respiratory resistive loads. In addition, the decreased number of co-labeled c-Fos and

GlyT2 neurons suggests that activation of glycinergic neurons in the cNTS was

abolished by bilateral cervical vagotomy but were only partially suppressed in the rNTS.

The different degree of glycinergic neuron suppression between caudal and rostral parts

of the NTS may represent different interconnections between neurons in the brainstem

control network. Therefore, further research needs to be performed to differentiate the

roles of the rNTSl and cNTS in controlling breathing pattern during respiratory load

compensation.

62

Table 3-1. Comparisons of Ti, Te, Tt, Pes and EMGdia between different breaths of C, O and R phases in anesthetized animals with intact vagi. p- values for all pairwise combination of all conditions for Ti, Te, Tt, Pes and EMGdia. N/A represents not significant between groups.

Ti Te Tt Pes EMGdia

C1 vs. O1 <.05 <.05 <.05 <.05 N/A

C1 vs. O2 N/A <.05 <.05 <.05 N/A

C1 vs. O3 N/A 0.05 <.05 <.05 N/A

C1 vs. R1 N/A <.05 N/A N/A <.05

C1 vs R2 N/A N/A N/A N/A N/A

C1 vs. R3 N/A <.05 N/A N/A <.05

O1 vs. R1 <.05 <.05 <.05 N/A N/A

O1 vs. R2 <.05 <.05 <.05 N/A N/A

O1 vs. R3 N/A <.05 <.05 <.05 N/A

O2 vs. R1 0.05 <.05 <.05 <.05 N/A

O2 vs. R2 <.05 <.05 <.05 N/A N/A

O2 vs. R3 N/A <.05 <.05 <.05 N/A

O3 vs. R1 N/A <.05 <.05 N/A N/A

O3 vs. R2 N/A <.05 <.05 N/A N/A

O3 vs. R3 N/A <.05 <.05 <.05 N/A

63

Table 3-2. Comparisons of Ti, Te, Tt, Pes and EMGdia between different breaths of C, O and R phases in vagotomized animals. p- values for all pairwise combination of all conditions for Ti, Te, Tt, Pes and EMGdia. N/A represents not significant between groups

Ti Te Tt Pes EMGdia

C1 vs. O1 N/A <.05 <.05 <.001 N/A

C1 vs. O2 N/A N/A N/A <.01 N/A

C1 vs. O3 N/A N/A N/A <.01 N/A

C1 vs. R1 N/A <.05 <.01 N/A N/A

C1 vs. R2 N/A <.001 <.05 N/A N/A

C1 vs. R3 N/A <.01 <.01 N/A N/A

O1 vs. R1 N/A N/A N/A <.001 N/A

O1 vs. R2 N/A N/A N/A <.01 N/A


O2 vs. R1 N/A N/A N/A <.001 N/A



O3 vs. R1 N/A N/A N/A <.001 N/A



64

Figure 3-1. Effect of vagotomy on the respiratory load compensation responses. Physiological results during control (C3-C1), occluded (O1-O3) and recovery (R1-R3) breaths. Top panel: breathing pattern in a rat with intact vagi. Bottom panel: breathing pattern in a rat after bilateral cervical vagotomy. The Ti and Te are shown in the trace of integrated EMGdia. Shadow area represents the period of tracheal occlusion

65

A B

C

Figure 3-2. ETTO elicited load compensation responses in anesthetized animals with

intact vagi. Normalized breath timing values during control (C3, C2, C1), occluded (O1, O2, O3) and recovery (R1, R2, R3). (A) The relationship between Ti and breath number. (B) The relationship between Te and breath number. (C) The relationship between Ttot and breath number. The * indicates a significant difference, p < 0.05

Breath

C3 C2 C1 O1 O2 O3 R1 R2 R3

Insp

iraotr

y T

ime (

Rela

tive U

nits

)

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

*

Breath

C3 C2 C1 O1 O2 O3 R1 R2 R3

Exp

iraotr

y T

ime (

Rela

tive U

nits

)

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

*

*

*

*

Breath

C3 C2 C1 O1 O2 O3 R1 R2 R3

Tota

l B

reath

Tim

e (

Rela

tive

Units)

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

*

*

*

66

A B

Figure 3-3. ETTO effect on EMGdia activity and Pes in anesthetized animals with intact vagi. Normalized EMGdia (A) and Pes (B) during control (C3, C2, C1), occluded (O1, O2, O3) and recovery (R1, R2, R3) breaths. The * indicates a significant difference, p < 0.05

Breath

C3 C2 C1 O1 O2 O3 R1 R2 R3

EM

Gdia

(R

ela

tive U

nits

)

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

H

Breath

C3 C2 C1 O1 O2 O3 R1 R2 R3

Eso

phageal P

ress

ure

(R

ela

tive

Units)

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

*

*

*

67

A B

C

Figure 3-4. Vagotomy load compensation responses with ETTO in anesthetized animals. Normalized breath timing values during control (C3, C2, C1), occluded (O1, O2, O3) and recovery (R1, R2, R3). (A) The relationship between Ti and breath number. (B) The relationship between Te and breath number. (C) The relationship between Ttot and breath number. The * indicates a significant difference, p < 0.05

Breath

C3 C2 C1 O1 O2 O3 R1 R2 R3

Inspira

tory

Tim

e (

Re

lative U

nits)

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

.

Breath

C3 C2 C1 O1 O2 O3 R1 R2 R3

Expirato

ry T

ime (

Rela

tive U

nits)

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

*

Breath

C3 C2 C1 O1 O2 O3 R1 R2 R3

Tota

l B

reath

Tim

e (

Re

lative U

nits)

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

*

68

A B

Figure 3-5. ETTO effect on Pes and EMGdia in vagotomized animals. Normalized Pes (A) and EMGdia (B) during control (C3, C2, C1), occluded (O1, O2, O3) and recovery (R1, R2, R3) breaths. The * indicates a significant difference, p<0.05; the ** indicates a significant difference, p < 0.01; the *** indicates a significant difference, p < 0.001

,

Breath

C3 C2 C1 O1 O2 O3 R1 R2 R3

Eso

phageal P

ress

ure

(R

ela

tive U

nits

)

0.5

1.0

1.5

2.0

2.5

3.0

*** ** **

Breath

C3 C2 C1 O1 O2 O3 R1 R2 R3

EM

Gd

ia (

Re

lative U

nits)

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

.

69

Figure 3-6. ETTO activated inhibitory glycinergic neurons in the cNTS in anesthetized animals with intact vagi. Immunofluorescence double-staining of c-Fos (red) and GlyT2 (green) in the cNTS (Bregma -13.8 mm) in animals without ETTO (B-G) and with ETTO (H-M). The dashed line in part A represents the area of rat brain atlas corresponding to the region in B-D and H-J. E-G and K-M represent the dashed area in B-D and H-J respectively. TS: Solitary Tract. Arrows represent immunoreactive cells. Photo courtesy of Hsiu-Wen Tsai

A

H I J

K L

TSTSTS

B C D

E F G

TS TSTS

c-Fos GlyT2 Merge

Intact vagi

Intact vagi+ ETTO M

70

Figure 3-7.The effect of vagotomy on the activation of inhibitory glycinergic neurons in the cNTS in anesthetized animals. Immunofluorescence double-staining of c-Fos (red) and GlyT2 (green) in the cNTS (Bregma -13.8 mm) in vagotomized animals without ETTO (B-G) and with ETTO (H-M). The dashed line in part A represents the area of rat brain atlas corresponding to the region in B-D and H-J. E-G and K-M represent the dashed area in B-D and H-J respectively. TS: Solitary Tract. Arrows represent immunoreactive cells. Photo courtesy of Hsiu-Wen Tsai

A

H I J

K L

TSTS

TS

B C D

E F G

TS TSTS

c-Fos GlyT2 Merge

Vagotomy

Vagotomy+ ETTO

M

71

A B

C

Figure 3-8. Immunofluorescence double-staining of c-Fos and GlyT2 in the cNTS in

anesthetized animals with or without intact vagi. (A) c-Fos labeled cell number (B) co-labeled c-Fos and GlyT2 cell number (C) the percentage of c-Fos positive cells co-labeled with GlyT2. The * indicates a significant difference, p < .05; the ** indicates a significant difference, p < 0.01; the *** indicates a significant difference, p < 0.001

.

Groups

Intact vagi

Intact vagi +

ITTO

Vagotomy

Vagotomy+ITTO

c-F

os

labele

d c

ell

num

ber

0

200

400

600

800

1000

Groups

Intact vagi

Intact vagi +

ITTO

Vagotomy

Vagotomy+ITTO

c-F

os

and G

lyT

2 c

ola

bele

d c

ell

num

ber

0

200

400

600

800

1000

** **

**

Groups

Intact vagi

Intact vagi +

ITTO

Vagotomy

Vagotomy+ITTO

c-F

os a

nd G

lyT

2 c

ola

bele

d c

ell

num

ber

/ c-F

os la

ble

d c

ell

num

ber

0

20

40

60

80

100

*** **

**

72

Figure 3-9. ETTO activated inhibitory glycinergic neurons in the rNTS in anesthetized animals with intact vagi. Immunofluorescence double-staining of c-Fos (red) and GlyT2 (green) in the rNTS (Bregma -13.2 mm) in animals without ETTO (B-G) and with ETTO (H-M). The dashed line in part A represents the area of rat brain atlas corresponding to the region in B-D and H-J. E-G and K-M represent the dashed area in B-D and H-J respectively. TS: Solitary Tract. Arrows represent immunoreactive cells. Photo courtesy of Hsiu-Wen Tsai

A

c-Fos GlyT2 Merge

Intact vagi

Intact vagi+ ETTO

B C D

E F G

H I J

K L M

TS TSTS

TSTS

TS

73

Figure 3-10. Effect of vagotomy on the activation of inhibitory glycinergic neurons in the rNTS in anesthetized animals. Immunofluorescence staining of c-Fos (red) and GlyT2 (green) in the rNTS (Bregma -13.2 mm) in vagotomized animals without ETTO (B-G) and with ETTO (H-M). The dashed line in part A represents the area of rat brain atlas corresponding to the region in B-D and H-J. E-G and K-M represent the dashed area in B-D and H-J respectively. TS: Solitary Tract. Arrows represent immunoreactive cells. Photo courtesy of Hsiu-Wen Tsai

A

c-Fos GlyT2 Merge

Vagotomy

Vagotomy+ ETTO

B C D

E F G

H I J

K L M

TS TSTS

TSTS

TS

74

A B

C

Figure 3-11. Immunofluorescence double-staining of c-Fos and GlyT2 in the rNTS in anesthetized animals with intact vagi and vagotomy. (A) c-Fos labeled cell number (B) co-labeled c-Fos and GlyT2 cell number (C) the percentage of c-Fos positive cells co-labeled with GlyT2. The * indicates a significant difference, p < 0.05

.

Groups

Intact vagi

Intact vagi +

ITTO

Vagotomy

Vagotomy+ITTO

c-F

os

labele

d c

ell

num

ber

0

200

400

600

800

1000

Groups

Intact vagi

Intact vagi +

ITTO

Vagotomy

Vagotomy+ITTO

c-F

os

and G

lyT

2 c

ola

bele

d c

ell

num

ber

0

200

400

600

800

1000

* *

p = 0.111

Groups

Intact vagi

Intact vagi +

ITTO

Vagotomy

Vagotomy+ITTO

c-F

os a

nd G

lyT

2 c

ola

bele

d c

ell

num

ber

/ c-F

os la

ble

d c

ell

num

ber

0

20

40

60

80

100

*** ***

**

75

A B

C

Figure 3-12. Immunofluorescence double-staining of c-Fos and GlyT2 in the combined

rNTS and cNTS in anesthetized animals with intact vagi and vagotomy. (A) c-Fos labeled cell number. (B) co-labeled c-Fos and GlyT2 cell number. (C) the percentage of c-Fos positive cells co-labeled with GlyT2. The * indicates a significant difference, p < 0.05; the ** indicates a significant difference, p < .01; the *** indicates a significant difference, p < 0.001

,

Groups

Intact vagi

Intact vagi +

ITTO

Vagotomy

Vagotomy+ITTO

c-F

os

labele

d c

ell

num

ber

0

500

1000

1500

2000

Groups

Intact vagi

Intact vagi +

ITTO

Vagotomy

Vagotomy+ITTO

c-F

os

and G

lyT

2 c

ola

bele

d c

ell

num

ber

0

500

1000

1500

2000

*** **

*

Groups

Intact vagi

Intact vagi +

ITTO

Vagotomy

Vagotomy+ITTO

c-F

os a

nd G

lyT

2 c

ola

bele

d c

ell

num

ber

/ c-F

os la

ble

d c

ell

num

ber

0

20

40

60

80

100

*** ***

**

76

CHAPTER 4 THE EFFECT OF TRACHEAL OCCLUSION ON RESPIRATORY LOAD

COMPENSATION AND CHANGES IN NEURONS CONTAINING INHIBITORY NEUROTRANSMITTER IN THE NUCLEUS OF THE SOLITARY TRACT IN

CONSCIOUS RATS

Background

Respiratory load compensation has been extensively studied in anesthetized

animals. There are relatively few studies of load compensation in the conscious state

although consciousness and behavior play a critical role in the modulation of breathing

pattern when animals encounter breathing challenges.

The respiratory load compensation responses are characterized as Vt-T

relationships in anesthetized animals. Applying a single inspiratory or expiratory load

decreased Vi and Ve and resulted in a prolongation of Ti and Te, respectively; the

timing parameters were not affected during unloaded breaths (Clark and von Euler,

1972; Zechman et al., 1976). This is a vagal-dependent reflex and mediated by PSRs in

lungs and airways. PSRs respond to changes in lung volume or transmural pressure

across the airways and synapse with second order interneurons in the NTS that project

to the PRG and VRG to modify the breathing pattern (Davenport et al., 1981a;

Davenport et al., 1981b; Davenport and Wozniak, 1986). In addition to the Vt-T

relationship, respiratory load compensation is also characterized by an increase of

respiratory motor outputs, including diaphragm and abdominal muscle activity (Koehler

and Bishop, 1979; Lopata et al., 1983). In anesthetized animals, the load compensation

responses are mediated by the respiratory neurons in the brainstem.

In conscious animals, respiratory load compensation involves cortical (primary

sensorimotor cortices, supplementary motor and premotor cortex ) and subcortical

neural structures (thalamus, globus pallidus, caudate, cerebellum and limbic system) to

77

voluntarily modify breathing pattern (Davenport and Vovk, 2009). It has been

demonstrated that there is a gating system in the thalamus for the perception of

respiratory sensation (Chan and Davenport, 2008). The thalamus determines if a

respiratory load will be gated in or out of the cortical and subcorical regions according to

load intensity and duration. A gated in signal will be processed and integrated by the

limbic system (affective dimension) and somatosensory cortex (discriminative

dimension) (von Leupoldt and Dahme, 2005, 2007). The affective dimension is

responsible for the processing of the emotional component of the respiratory input

(Davenport and Vovk, 2009). The discriminative dimension is related to the

determination of the spatial, temporal and intensity perception of the respiratory inputs

(von Leupoldt and Dahme, 2005; von Leupoldt et al., 2008). The motor cortex receives

the integrated sensory information and sends projections to the brainstem or directly to

the motorneurons in the spinal cord to allow voluntarily modification of the reflexive

pattern of load compensation.

Conscious animals respond to respiratory load differently from anesthetized

animals. In conscious dogs, single tracheal occlusion at end expiration prolonged Ti and

decreased breathing frequency (Phillipson, 1974). Conscious newborn lambs

compensated to a single expiratory load by dilating the larynx and prolonging the Te,

followed by a return to baseline during the post-load breath (Watts et al., 1997).

Application of two consecutive external inspiratory loads to conscious goats caused

prolonged Ti and augmented respiratory output during the two loaded breaths (Hutt et

al., 1991). In awake ponies, sustained external inspiratory loads for 4 minutes resulted

in an increase in Ti, decrease in Te and augmentation of diaphragm activity during the

78

first loaded breath and only had minimal changes after first loaded breath (Forster et al.,

1994). In conscious humans, application of resistive loads for an entire breath resulted

in a prolongation of Ti, Te and Ttot (Calabrese et al., 1998). The conflicting results

between these studies may be due to different experimental designs, including

differences in load strengths, load durations, load applications, species and age.

Previous studies used external respiratory loads to elicit load compensation responses

and only applied the load to a single phase of respiration (inspiration or expiration) or a

single entire breath. To our knowledge, respiratory load compensation elicited by

sustained intrinsic tracheal occlusions in consciousness has not been studied. In our

laboratory, we developed a new surgical strategy to produce intrinsic, transient tracheal

occlusion without changing lung compliance. The first purpose of the present study is to

determine the ITTO-elicited load compensation responses in conscious animals.

Additionally, respiratory drive in anesthetized animal is mainly dominated by the

reciprocal interconnections within the brainstem neural network. In the model proposed

by Rybak (Rybak et al., 2007; Rybak et al., 2008), the brainstem respiratory neural

network consists of synaptic connections between the pons, ventral medulla and NTS.

Inhibitory reciprocal interconnections between the post-I, aug-E in the BötC and pre-I,

early-I in the pre- BötC are proposed to generate the respiratory rhythm. The pons has

excitatory projections to the respiratory neurons in the dorsal and ventral medulla to

modulate the rhythm and pattern of breathing. The NTS is the site of integrating and

processing respiratory peripheral afferents from lungs and airways such as PSRs,

RARs and C-fibers for further modification of breathing pattern to generate appropriate

ventilation for the body’s demand. PSRs in the lungs and airways are the major

79

receptors sensing the changes in lung volume or transmural pressure and synapse on

the p-cells in the NTS and project to other respiratory neurons in the medulla and pons

for the reflex control of breathing pattern (Davenport et al., 1981a). The NTS is critically

important for the neurogenesis of load compensation responses in anesthetized animals.

It has been demonstrated that glycinergic interneurons in the NTS were activated by

ITTO (Chapter 2) and ETTO (Chapter 3) in anesthetized animals. However, in

conscious animals, the breathing pattern is modulated by cortical and subcotical

structures implying that consciousness and behavior are important for the neurogenesis

of conscious load compensation responses. Therefore, the second purpose of this study

is to determine if the glycinergic neurons in the NTS are activated in the neurogenesis

of the load compensation responses in conscious animals. We hypothesized that

glycinergic neurons in the NTS would be less activated by ITTO in conscious animals

compared to anesthetized animals. We used immunofluorescence double staining of c-

Fos and GlyT2 to test our hypothesis.


Animals

Experiments were performed on 11 male Sprague-Dawley rats (320-380g). The

animals were housed in the University of Florida animal care facility. They were

exposed to a 12 hr light/12 hr dark cycle and with free access to food and water. The



Surgical Procedure

Animals were anesthetized using inhaled isoflurane gas (2-5% in O2). Adequate

anesthetic depth was verified by the absence of a withdrawal reflex to a rear paw pinch.

80

Buprenorphine (0.04-0.05 mg/kg body weight) and carprofen (5mg/kg body weight)

were administered preoperatively via subcutaneous injection. The eyes were coated

with petroleum ointment to prevent drying. Incision sites were shaved and sterilized with

povidine-iodine topical antiseptic solution. Body temperature was monitored with a

rectal probe and maintained at 37-39°C using a heating pad.

The trachea was exposed through a ventral incision. The trachea was separated from

surrounding tissue. An inflatable cuff (Fine Science Tools) was sutured around the

trachea, two cartilage rings caudal to the larynx. The cuff was connected to a saline-

filled syringe via a rubber tube. The rubber tube was routed subcutaneously to the

dorsal neck surface and externalized through an incision between the scapulae. The

tube was fixed in the skin using closing sutures. The ventral incision was closed using

an interrupter suture pattern. Following surgery, rats were administered normal saline

(0.01-0.02 ml/g body weight). Postoperative analgesia was provided with buprenorphine

(0.01-0.05 mg/kg body weight) and carprofen (5 mg/kg body weight) every 24 h for at

least three days. Animals were allowed a full week recovery before training protocol

began.


The Sprague-Dawley rats (320-380 g) were randomly divided into experimental (n=5)

and sham (n=6) groups. One week after the surgery, the rats were placed in the

restrainer for 3 hours per day for two days. On post-operative day10, experimental

group rats were placed in the restrainer for 90 min, followed by 10 minutes of ITTO then

90 min post-occlusion eupneic breathing in the restrainer; sham group rats were placed

in the restrainer for 3 hours without receiving ITTO. Following the time in the restrainer,

the animals were euthanized and perfused. Brains were harvested and fixed in 4%

81

paraformaldehyde for 24 h and then transfer into a solution of 30% sucrose in PBS. The

fixed tissue was coronally sectioned into 40 μm thick with a cryostat (HM101, Carl

Zeiss) for immunofluorescence analysis.


The double-staining methods are the same as described in Chapter 3.

Data Analysis

The data analysis methods are the same as described in Chapter 2.


The statistical analyses are the same as described in Chapter 2.

Results

Breathing Pattern

Figure 4-1 illustrates the breathing pattern in conscious animals. There were no

significant differences in Ti (Figure 4-2A) and EMGdia (Figure 4-3) between O, C and R

phases. Te was significantly prolonged during O3 but not O1 and O2 breaths when

compared to C and R phases (df = 8, F = 5.916, p < 0.001) (Table 4-1, Figure 4-2B).

Prolongation of Te contributed to the significant increase of Ttot during O2 and O3 when

compared to C and R phases (df = 8, F = 10.876, p < 0.001) (Table 4-1; Figure 4-3C).

There were no significant differences in Ti, Te, Ttot, EMGdia and Pes at matched time

points in the sham animals.

lmmunofluorescence Double-Staining

In the cNTS, there were no significant differences in the number of c-Fos cells, co-

labeled c-Fos and GlyT2 cells and the percentage of c-Fos positive cells co-labeled with

GlyT2 between experimental and sham groups (Figure 4-4, Figure 4-5).

82

In the rNTS (Figure 4-6), there were no significant difference in the number of c-Fos

cells and co-labeled c-Fos and Gly T2 cells between these two groups (Figure 4-7A,

Figure 4-7B). The percentage of c-Fos positive cells co-labeled with GlyT2 showed

significant differences between two groups (df = 1, F= 12.647, p < 0.01) (Figure 4-7C).

The percentage of c-Fos positive cells co-labeled with GlyT2 was significantly greater in

the experimental group than the sham group (t(9) = 3.562, p < 0.01 ).

In the combined rNTSl and cNTS, the number of c-Fos cells showed no significant

difference between experimental and sham groups (Figure 4-8A). There was trend for

increased number of co-labeled c-Fos and GlyT2 cells (df = 1, F = 3.830, p = 0.082)

(Figure 4-8B). There was a significant difference in the percentage of c-Fos positive

cells co-labeled with GlyT2 between two groups (df = 1, F = 9.544, p < 0.05). The

percentage of c-Fos positive cells co-labeled with GlyT2 was significantly greater in the

experimental group than the sham group (t(9) = 3.089, p < 0.05) (Figure 4-8C).

Discussion

Breathing Pattern

The load compensation responses in conscious animals were found to be different

from those in anesthetized animals. In anesthetized animals, ITTO resulted in

prolongation of Ti, Te and Ttot which is reflex load compensation mediated by the

brainstem. However, conscious animals behaviorally prolonged Te in response to ITTO

without changing Ti. The prolongation of Te contributes to the increase of Ttot. During

occlusions, conscious rats did not respond to ITTO in the beginning of the occlusions

(O1 and O2), but behaviorally increased their expiration only by O3 and then returned to

normal breathing immediately after the removal of occlusions demonstrating that the

load compensation response in conscious animals might be affected by consciousness

83

and behavior. These results are different from previous studies (Calabrese et al., 1998;

Forster et al., 1994; Hutt et al., 1991; Phillipson, 1974; Watts et al., 1997) that have only

used single breath loads during one phase of breathing (inspiration or expiration) or one

entire breath. However, in the present study, consecutive sustained respiratory loads to

both inspiration and expiration were presented in conscious animals for up to 2-3 sec

that could cause an aversive sensation. If an aversive sensation was elicited with the

ITTO then the conscious animals may hold their breaths during late phase of ITTO

rather than try to breathe against the loads, i.e. behavior load compensation. In addition,

ITTO was used to elicit load compensation responses in this study whereas previous

studies used external respiratory loads to generate load compensation. In fact, ITTO is

a more appropriate method to simulate patients with respiratory obstructive diseases

compared to external respiratory load since it reduces airflow intrinsically without

changing lung compliance.


ITTO elicited the activation of glycinergic neurons in the rNTS but not in the cNTS in

conscious animals. This is in contrast with the results, in anesthetized animals with

ITTO (Chapter 2) and ETTO (Chapter 3), in which glycinergic neurons were activated in

both rostral and caudal parts of NTS. These results support our hypothesis that the NTS

plays a reduced role for the neurogenesis of load compensation in conscious animal

compared to anesthetized animals. Furthermore, ITTO-activated glycinergic neurons

were concentrated in the SolM, SolV and SolVL. Glycinergic neurons activated in the

SolM and SolV is consistent with the previous studies that ITTO and ETTO activated

glycinergic neurons in the same subdivisions of NTS in anesthetized animals. The

activation of glyciergic neurons in the SolVL was only seen in conscious animals but not

84

in anesthetized animals. It has been reported that application of excitatory amino acids

on the SolM containing p-cells caused reflex termination of inspiration and prolongation

of expiration, while blockade of excitatory amino acid in this area reduced these

changes (Bonham et al., 1993; Bonham and McCrimmon, 1990). However, the SolVL is

not critical for production of these respiratory reflexes since deletion of this area still

leaves these reflexes intact (McCrimmon et al., 1987). In addition, previous studies

have shown that neurons in the SolM mainly project to the ventrolateral medulla,

parafacial region and rVRG and only few SolM neurons send projections to the BötC

and pre-BötC (Alheid et al., 2011). The projections from SolVL to the VRC are more

broadly distributed compared to other subdivisions of NTS (Alheid et al., 2011).

Therefore, the activated glycinergic neurons in the SolM in both conscious and

anesthetized animals may be the primary neural area responsible for the generation of

load compensation responses. Additionally, the different distribution of projections from

the SolM and SolVL might mediate difference mechanisms to modulate breathing

pattern.

In the present study, activated glycinergic neurons in the SolM and SolV may be

vagally mediated p-cells. P-cells are the second order neurons in the NTS activated by

PSRs in the lungs and airways and send projection to the VRC and pons for the

modulation of breathing pattern. It has been demonstrated that two-thirds of p-cells in

the NTS use GABA and glycine as neurotransmitters (Ezure and Tanaka, 2004).

Although we saw the elevated expression of glycinergic neurons in the SolM and SolV

in conscious animals as we found in anesthetized animals, the patterns of load

compensation responses are different between conscious and anesthetized states

85

suggesting that other mechanisms play more important roles in the behavioral load

compensation. SolVL is a candidate brain region for the neurogenesis of load

compensation in conscious animals since glycinergic neurons in the SolVL are only

activated in conscious animals. As noted previously, the SolVL is not necessary for

reflex load compensation. Several studies have demonstrated that the SolVL receives

projections from cortical and subcortical structures, such as subcortical cortex (van der

Kooy et al., 1984) and the central nucleus of amygdala (Danielsen et al., 1989). The

activated glycinergic neurons in the SolVL may be activated by these higher cortical and

subcortical structures for behaviorally control of breathing pattern.

To our knowledge, this is the first study to investigate the importance of glycinergic

neurons in the NTS for the neurogenesis of load compensation responses with

sustained respiratory loads in conscious animals. Based on these results, cortical and

subcortical processing of respiratory load sensation overrides brainstem reflexes for

conscious modification of breathing pattern.

86

Table 4-1. Comparisons of Ti, Te, Tt, Pes and EMGdia between different breaths of C, O and R phases in conscious animals. p- values for all pairwise combination of all conditions for Ti, Te, Tt, Pes and EMGdia. N/A represents not significant between groups

Ti Te Tt EMGdia

C1 vs. O1 N/A N/A N/A N/A

C1 vs. O2 N/A N/A <.05 N/A

C1 vs. O3 N/A <.05 <.05 N/A

C1 vs. R1 N/A N/A N/A N/A

C1 vs R2 N/A N/A N/A N/A

C1 vs. R3 N/A N/A N/A N/A

O1 vs. R1 N/A N/A N/A N/A

O1 vs. R2 N/A N/A N/A N/A

O1 vs. R3 N/A N/A <.05 N/A

O2 vs. R1 N/A N/A <.05 N/A

O2 vs. R2 N/A N/A <.001 N/A

O2 vs. R3 N/A N/A <.05 N/A

O3 vs. R1 N/A N/A <.05 N/A

O3 vs. R2 N/A N/A <.01 N/A

O3 vs. R3 N/A N/A <.01 N/A

87

Figure 4-1. Sustained ITTO behavior modulation of breathing pattern in conscious animals. Physiological results during control (C3-C1), occluded (O1-O3) and recovery (R1-R3) breaths. Determination of Ti and Te are shown in the trace of integrated EMGdia. Shadow area represents the period of tracheal occlusions

Tracheal occluder

Pressure (mmHg)

EMGdia

Integrated EMGdia

1 2 3 4 5 6 7 8 9 10Time (sec)

TiTe

Occlusions

C3 C2 C1 O1 O2 O3 R1 R2 R3

88

A B

C

Figure 4-2. Effect of sustained ITTO in conscious animals. Normalized breath timing values during control (C3, C2, C1), occluded (O1, O2, O3) and recovery (R1, R2, R3). (A) The relationship between Ti and breath number. (B) The relationship between Te and breath number. (C) The relationship between Ttot and breath number. The * indicates a significant difference, p < 0.05

Breath

C3 C2 C1 O1 O2 O3 R1 R2 R3

Insp

iraoto

ry T

ime (

Rela

tive U

nits

)

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

>

Breath

C3 C2 C1 O1 O2 O3 R1 R2 R3

Exp

irato

ry T

ime (

Rela

tive U

nits

)

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

*

Breath

C3 C2 C1 O1 O2 O3 R1 R2 R3

Tota

l Bre

ath

Tim

e (

Rela

tive U

nits

)

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

*

*

89

Figure 4-3. Sustained ITTO effect on EMGdia in conscious animals. Normalized EMGdia during control (C3, C2, C1), occluded (O1, O2, O3) and recovery (R1, R2, R3) breaths in a conscious animal with ITTO

Breath

C3 C2 C1 O1 O2 O3 R1 R2 R3

EM

Gdia

(R

ela

tive U

nits

)

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

,

90

Figure 4-4. The effect of ITTO on the activation of inhibitory glycinergic neurons in the cNTS in conscious animals. Immunofluorescence double staining of c-Fos (red) and GlyT2 (green) in the cNTS (Bregma -13.8 mm) in sham (B-G) and experimental groups (H-M). The dashed line in part A represents the area of rat brain atlas corresponding to the region in B-D and H-J. E-G and K-M represent the dashed area in B-D and H-J, respectively. TS: Solitary Tract. Arrows represent immunoactive cells. Photo courtesy of Hsiu-Wen Tsai

A

H I J

K L

TSTS TS

B C D

E F G

TS TSTS

c-Fos GlyT2 Merge

Sham

MExp

91

A B

C

Figure 4-5. Immunofluorescence double-staining of c-Fos and GlyT2 in the cNTS in

conscious animals. (A) c-Fos labeled cell number (B) co-labeled c-Fos and GlyT2 cell number (C) the percentage of c-Fos positive cells co-labeled with GlyT2

;

Groups

Sham Exp

c-F

os la

bele

d c

ell

num

ber

0

200

400

600

800

1000

;

Groups

Sham Exp

c-F

os a

nd G

lyT

2 c

ola

bele

d c

ell

num

ber

0

200

400

600

800

1000

;

Groups

Sham Exp

c-F

os a

nd

Gly

T2

co

lab

ele

d c

ell

nu

mb

er

/ c-F

os la

be

led

ce

ll n

um

be

r

0

20

40

60

80

100

92

Figure 4-6. ITTO activated inhibitory glycinergic neurons in the rNTS in conscious animals. Immunofluorescence double-staining of c-Fos (red) and GlyT2 (green) in the rNTS (Bregma -13.2 mm) in sham (B-G) and experimental groups (H-M). The dashed line in part A represents the area of rat brain atlas corresponding to the region in B-D and H-J. E-G and K-M represent the dashed area in B-D and H-J, respectively. TS: Solitary Tract. Arrows represent immunoactive cells. Photo courtesy of Hsiu-Wen Tsai

A

c-Fos GlyT2 Merge

Sham

Exp

B C D

E F G

H I J

K L M

TS TSTS

TSTS

TS

93

A B

C

Figure 4-7. Immunofluorescence double-staining of c-Fos and GlyT2 in the rNTS in

conscious animals. (A) c-Fos labeled cell number (B) co-labeled c-Fos and GlyT2 cell number (C) the percentage of c-Fos positive cells co-labeled with GlyT2. The ** indicates a significant difference, p < 0.01

,

Groups

Sham Exp

c-F

os

labele

d c

ell

num

ber

0

200

400

600

800

1000

Groups

Sham Exp

c-F

os a

nd G

lyT

2 c

ola

bele

d c

ell

num

ber

0

200

400

600

800

1000

,

Groups

Sham Exp

c-F

os a

nd

Gly

T2

co

lab

ele

d c

ell

nu

mb

er

/ c-F

os la

be

led

ce

ll n

um

be

r

0

20

40

60

80

100

**

94

A B

C

Figure 4-8. Immunofluorescence double-staining of c-Fos and GlyT2 in the combined

rNTS and cNTS in conscious animals. (A) c-Fos labeled cell number (B) co-labeled c-Fos and GlyT2 cell number (C) the percentage of c-Fos positive cells co-labeled with GlyT2. The * indicates a significant difference, p < 0.05

Groups

Sham Exp

c-F

os

labele

d c

ell

num

ber

0

500

1000

1500

2000

,

Groups

Sham Exp

c-F

os a

nd G

lyT

2 c

ola

bele

d c

ell

num

ber

0

500

1000

1500

2000

p = 0.082

Groups

Sham Exp

c-F

os a

nd G

lyT

2 c

ola

bele

d c

ell

num

ber

0

500

1000

1500

2000

*

95

CHAPTER 5 THE IMPACT OF EMOTION ON THE PERCEPTION OF GRADED MAGNITUDES OF

RESPIRATORY RESISTIVE LOADS

Background

Breathing is a continuous process and usually not sensed under normal conditions.

However, patients with respiratory diseases such as chronic obstructive pulmonary

disease (COPD) and asthma commonly have increased loads to the ventilatory muscles

due to increased stiffness of the lungs or increased resistance in the airways. With

increased respiratory loads, their ventilatory muscles increase muscle force to

compensate for the increased load eliciting the conscious sensations of breathing effort

(Altose et al., 1985; Davenport and Vovk, 2009).

Mechanical receptors located in the inspiratory muscles (i.e. the intercostals and

diaphragm) are the major afferents that project to higher brain centers to elicit

respiratory sensations of breathing effort. Respiratory sensations are hypothesized to

originate from two neural pathways, discriminative processing and affective processing.

Discriminative processing is relayed to the somatosensory network and determines the

spatial, temporal and intensity perception of the respiratory input (Davenport and Vovk,

2009). Affective processing is relayed to components of the limbic system (i.e. the

insular and cingulate cortices) that evaluate the emotional component of the respiratory

input (von Leupoldt and Dahme, 2005; von Leupoldt et al., 2008).

Adequate perception of respiratory sensations is important for motivating patients to

seek timely medical care. Reduced respiratory perception in respiratory disease might

delay the initiation of treatment resulting in an increase in morbidity (Barnes, 1994;

Feldman et al., 2007; Julius et al., 2002; Magadle et al., 2002). However, over-

perception of respiratory sensations might also be a problem by causing overuse of

96

medications or avoidance of physical activity (Main et al., 2003; Ng et al., 2007; von

Leupoldt and Dahme, 2007).

A growing body of research suggests that emotion affects the perception of

respiratory sensations regardless of physiological ventilatory changes. These studies

have shown that patients with COPD or asthma with high negative affectivity (NA)

reported more enhanced symptoms compared to patients with lower NA, without

corresponding differences of respiratory function (De Peuter et al., 2008; Vogele and

von Leupoldt, 2008). In addition, patients and healthy individuals also showed increased

reports of respiratory sensations during experimentally induced, short lasting negative

affective states (Affleck et al., 2000; Bogaerts et al., 2005; von Leupoldt et al., 2006b;

von Leupoldt et al., 2008; von Leupoldt et al., 2010). For example, sustained breathing

through respiratory resistive loads resulted in greater reports of breathing effort when

patients with asthma and COPD as well as healthy volunteers viewed emotionally laden

pictures and videos of unpleasant compared to neutral or pleasant content. External

respiratory resistive loads increase the resistive work of breathing and are commonly

used to study respiratory load afferent inputs to the central nervous system, cognitive

perception of increased work of breathing and motor load compensation outputs (Axen

and Haas, 1979; Davenport et al., 2007; Puddy et al., 1992; Webster and Colrain, 2000).

However, the effect of different emotional states on the perception of graded

magnitudes of respiratory resistive loads has not been investigated. Thus, it remains

unknown whether emotional modulation of respiratory perception is different at different

levels of respiratory restriction.

97

The purpose of the present study was to investigate the role of emotion on the

magnitude estimation (ME) of inspiratory resistive loads that induce five different levels

of breathing effort. The participants’ emotional states were modulated using the

established method of affective picture viewing (Lang, 2008).


Participants

The study was approved by the Institutional Review Board of the University of

Florida. Twenty-four healthy adults with no history of pulmonary or neurological disease

participated in the study after providing informed written consent. To ensure vital

baseline lung function, participants underwent standard spirometry prior to testing.

Baseline characteristics of the participants are listed in Table 5-1.

Affective Picture Series

Two hundred and sixteen pictures were selected from the International Affective

Picture System (IAPS) (Lang, 2008). Based upon normative ratings, the pictures were

grouped into pleasant, neutral and unpleasant affective categories, each consisting of

72 pictures. Each category was further divided into 2 series of 36 pictures. Each picture

in the 6 affective picture series was presented on a monitor for 10 sec resulting in a total

of 6 min of picture presentation time for each series.

Inspiratory Resistive Loads

The resistive loading manifold was placed at the distal part of a breathing circuit and

consisted of 5 magnitudes of resistive loads (5, 10, 15, 20, 45 cmH2O/L/sec) and no

load. Resistive loads were sintered bronze disks placed in series in the loading manifold

and separated by stoppered ports. The resistances were presented for a single

inspiration by removing a stopper from one selected port and allowing the subject to

98

inspire through the load. Following each load presentation, participants estimated the

perceived difficulty of breathing using the modified Borg scale which ranges from 0 (=

nothing at all) to 10 (=very, very serve) (Borg, 1982).

Measurement of Respiration

Mouth pressure was measured from a port in the center of the nonrebreathing valve

with a differential pressure transducer. Inspiratory airflow was also measured with a

differential pressure transducer connected to the pneumotachograph at the inspiratory

port of the nonrebreathing valve by reinforced tubing. All signals were stored and

digitized using a PowerLab data acquisition system (ADInstruments, Colorado Springs,

CO, USA).

Measurement of Emotional Responses

The participants were asked to rate their emotional state on the dimensions of

valence and arousal after each picture series with a paper and pencil version of the

Self-Assessment Manikin (SAM) (Bradley and Lang, 1994). The ratings of valence and

arousal range from 1(=low) to 9 (=high).

Procedure

The experimental set-up is illustrated in Figure 5-1. The five resistive inspiratory

loads and no-load were presented in a randomized block design. Two blocks were

presented in each picture series with each block containing one presentation of the five

loads and no load in random order. A total of 4 presentations of each load magnitude

and no load were presented during each of the 3 affective conditions (pleasant, neutral

and unpleasant). The participants were seated comfortably in a reclining lounge chair.

They breathed through a mouth piece connected to a nonrebreathing valve. The

inspiratory port of the nonrebreathing valve was connected to a pneumotachograph and

99

resistive loading manifold by reinforced tubing. The participants were instructed that

when a light on the top of the monitor was illuminated, a resistive load would be applied

to the next inspiration. Following the loaded breath, they estimated the perceived

difficulty of breathing load using the modified Borg scale. At the end of each series,

participants provided ratings of valence and arousal.


Valence and arousal ratings were analyzed with separate one-way repeated

measures analyses of variance (ANOVA) with condition (pleasant, neutral, unpleasant)

as factor which were followed up by post-hoc paired t-tests. ME ratings were averaged

across the four presentations of each load for each affective condition and logarithmized

(LogME). Pmax, airflow and LogME were analyzed using two-way repeated measures

ANOVAs with factors condition (pleasant, neutral, and unpleasant) and loads (5 graded

resistive loads) which were followed up by post-hoc paired t-tests. The slope of LogME-

LogPmax was calculated as a measure of the sensitivity to the respiratory loads (Kelsen

et al., 1981; Kifle et al., 1997; Killian et al., 1981). A one-way repeated measures

ANOVA with condition as factor was used to analyze the slope of LogME- LogPmax

relationship which was followed up by post-hoc paired t-tests. A Greenhouse-Geisser

correction was applied in case of violated sphericity assumptions with reported

significance levels referring to corrected degrees of freedom. The significance criterion

for all analyses was set at p < 0.05.

Results

Ratings of valence showed significant differences between the three affective

conditions (Figure 5-2) with significant decreases from the pleasant to neutral to

unpleasant picture series (df = 2, F = 73.78, p < 0.001, ε = .72) (pleasant vs. neutral:

100

t(23) = 6.20, p < 0.001; unpleasant vs. neutral: t(23) = 8.41, p < 0.001; unpleasant vs.

pleasant: t(23) = 9.50, p < 0.001).

Arousal ratings showed significant differences between the conditions (df = 2, F =

29.97, p < 0.001) with higher ratings for pleasant and unpleasant picture series

compared to the neutral series (pleasant vs. neutral: t(23) = 5.93, p < 0.001; unpleasant

vs. neutral: t(23) = 6.64, p < 0.001; unpleasant vs. pleasant: t(23) = 2.45, p < 0.001).

There were no statistically significant differences in airflow and mouth pressure

between the three affective conditions (Figure 5-3) indicating that the resistive loaded

breathing pattern was comparable across affective conditions.

The relationship between magnitude estimation of breathing difficulty and load

intensity during the different affective conditions is presented in Figure 5-4. A main

effect for load demonstrated that the LogME increased with increasing resistance load

magnitude in all three conditions (df = 4, F = 113.65, p < 0.001). An interaction effect

was observed between load and condition (df =8, F = 2.441, p < 0.05). Post-hoc test

showed that for the 5 cmH2O/L/sec load, the LogME was significantly higher during the

unpleasant picture series compared to the neutral and pleasant picture series,

(unpleasant vs. neutral: t(22) = 2.46, p < 0.05; unpleasant vs. pleasant: t(22) = 2.42, p <

0.05; pleasant vs. neutral: t(22) = 0.53, p > 0.05). However, for resistive loads greater

than 5 cmH2O/L/sec, no significant differences in the reported LogME were observed

between the three affective conditions. In addition, the slope of the LogME-LogPmax

showed a significant difference between the three affective conditions (df = 2, F = 3.32,

p < 0.05) with LogME-LogPmax for the unpleasant state being significantly lower than

101

for the neutral state (Figure 5-5) (unpleasant vs. neutral: t(23) = 2.56, p < 0.05; pleasant

vs. neutral: t(23) = -1.05, p > 0.05; unpleasant vs. pleasant: t(23) = 1.52, p > 0.05)

Discussion

The results demonstrated decreased ratings of valence from the pleasant to neutral

to unpleasant affective condition. Ratings of arousal were increased during the pleasant

and unpleasant conditions compared to the neutral condition. This pattern suggests that

different emotional states were successfully induced by viewing affective pictures series

which replicates previous studies using IAPS picture material (Bradley 2007; Van Diest

et al., 2009).

The reported magnitude estimation of resistive loads was only increased during the

unpleasant affective condition compared to the pleasant and neutral conditions, but only

at a load of 5 cmH2O/L/sec. With resistive loads greater than 5 cmH2O/L/sec, no

significant differences in the reported magnitude estimation were observed between the

three affective conditions. In addition, the slope of LogME-LogPmax during the

unpleasant affective state was significantly lower than during the neutral state, mainly

due to increases of LogME-LogPmax at the 5 cmH2O/L/sec load. This decrease in slope

is consistent with a decreased sensitivity of participants’ respiratory perception to

inspiratory respiratory loads during an unpleasant affective state compared to a neutral

state. No statistically significant differences were observed in airflow and mouth

pressure between the affective conditions which indicates that the impact of emotion on

magnitude estimation of resistive loads was the result of a difference in load perception

neural processes and not due to differences in breathing patterns. Taken together, the

results demonstrate a relationship between the impact of induced emotional states on

the perception of respiratory effort and the magnitude of resistive loads.

102

Our results are in accordance with previous studies that have reported negative

emotional state to increase the level of perceived breathing effort or respiratory

sensations in individuals with and without respiratory disease (Lavietes et al., 2000; Put

et al., 2004; Rietveld and Prins, 1998; von Leupoldt et al., 2006a; von Leupoldt et al.,

2006b). For example, Livermore and colleagues (2008) demonstrated that COPD

patients with comorbid panic attacks or panic disorders reported higher breathing effort

during resistive load breathing than those patients without panic attacks or panic

disorders despite comparable respiratory limitations (Livermore et al., 2008). Moreover,

in healthy participants, the ratings of the affective dimension of perceived breathing

effort during resistive load breathing were increased during unpleasant compared to

neutral or pleasant affective visual stimulation (von Leupoldt et al., 2006a; von Leupoldt

et al., 2008). However, these previous studies used only one level of respiratory

resistive load. The present results extend these previous findings by suggesting that this

emotional impact on the perception of respiratory effort is particularly prominent during

loads of rather small magnitude. This supports recent hypotheses claiming that the

emotional impact on respiratory symptom perception is particularly prominent when the

respiratory sensory signal is ambivalent, i.e. of low magnitude (Janssens et al., 2009). It

might be speculated that low resistive loads do not cause a pronounced aversive

sensation so the perception of respiratory effort might be mainly influenced by the

affective state. High resistive loads might cause a more significant aversive sensation

that, in turn, might override the impact of affective state on the perception of respiratory

effort and prevent a further modulation by affective state. This might also explain the

few findings of other studies demonstrating that negative emotionality reduces or has no

103

effect on the perception of respiratory sensations in patients with respiratory diseases

(Hudgel et al., 1982; Tiller et al., 1987; von Leupoldt et al., 2006b). The difference

between these reports may be a function of the magnitude of the employed increased

loads. Our results suggest there is a load threshold for affective state modulation of

respiratory perception.

However, the present results also differ from previous studies because these studies

could still demonstrate an emotional modulation of respiratory perception at higher

resistive load levels (> 30 cmH2O), which was not observed in the present data. This

discrepancy might be related to the fact that previous studies used sustained breathing

through the resistive loads of up to 3 min, whereas participants in the present study

provided magnitude ratings of loads that were presented for a single inspiration.

Therefore, it is possible that the emotional modulation of respiratory perception is a

complex combination of emotional state, magnitude of the respiratory stimulus and

duration of loaded breathing. Therefore, future studies are required to test the impact of

emotional state on the perception of different levels of resistive loads that are presented

in a sustained fashion.

The present findings underline that emotional factors play a critical role on respiratory

symptom perception. In patients with COPD and asthma, the level of respiratory

distress might be impacted by negative affective states rather than level of lung

function. Therefore, it is important to emphasize the detection and treatment of

prevalent affective disorders such as anxiety and depression in patients with respiratory

diseases in order to improve patients’ respiratory symptoms.

104

Table 5-1. Baseline characteristics of participants (Mean, SD)

Characteristics Data

Age (years) 28.1 (5.5) Sex (female/male) 14/10 Weight (kg) 64.8 (13.5) Height (cm) 169.2 (9.8) Forced expiratory volume in 1 s (L) 3.85 (0.92) Forced expiratory volume in 1 s (% of predicted value) 105.35 (12.08) Forced vital capacity (L) 4.52 (1.12) Forced vital capacity (% of predicted value) 106.02 (13.71) Forced expiratory volume in 1 s/Forced vital capacity (%) 85.63 (5.52)

105

Figure 5-1. Schematic of experimental setup

Resistance Manifold

PowerLab Data Acquisition Systems

Grass Polygraph

Load Indicator Light Load Indicator Switch

Borg Scale

Monitor

Inspiratory. Airflow

Mouth Pressure

SUBJECT ISOLATION ROOM

106

A

B

Figure 5-2. Mean SAM ratings of valence (a) and arousal (b). Evaluative ratings of

valence and arousal differed significantly following viewing of pleasant, neutral and unpleasant picture series, p < 0.001. Error bars represent standard errors of the mean. ***p < 0.001, **p < 0.001

Valence

Picture Series

Pleasant Neutral Unpleasant

Se

lf A

sse

ssm

en

t M

an

ikin

1

2

3

4

5

6

7

8

9

***

*** ***

Arousal

Picture Series


Se

lf A

sse

ssm

en

t M

an

ikin

1

2

3

4

5

6

7

8

9

*** ***

**

107

A

B

Figure 5-3. Peak mouth pressure Pmax (a) and peak airflow (b) at different levels of

load resistance during pleasant, neutral and unpleasant picture series. Error bars represent standard errors of the mean

Resistance (cmH2O/L/sec)

0 10 20 30 40 50

Mo

uth

Pre

ssure

(cm

H2O

)

-10

-8

-6

-4

-2

0

Pleasant

Neutral

UnpleasantR1

R2

R3

R4

R5

Resistance (cmH2O/L/sec)

0 10 20 30 40 50

Airflo

w (

L/m

in)

0.0

0.2

0.4

0.6

0.8

Pleasant

Neutral

Unpleasant

R1

R2 R3 R4

R5

108

Figure 5-4. The LogME–LogPmax relationship for the five resistive loads during

pleasant, neutral and unpleasant picture series. Error bars represent standard errors of the mean. *p < 0.05

Log of Mouth Pressure

0.4 0.6 0.8 1.0

Log o

f M

agnitude E

stim

ation

-0.2

0.0

0.2

0.4

0.6

0.8

1.0 Pleasant

Neutral

Unpleasant

*

R1

R2

R3

R4

R5

109

Figure 5-5. Mean of slope of LogME-LogPmax for the pleasant, neutral and unpleasant

picture series. Error bars represent standard errors of the mean. * p < 0.05

Picture Series


Slo

pe o

f Lo

gM

E-L

ogP

ma

x

0.0

0.5

1.0

1.5

2.0

2.5

*

110

CHAPTER 6 SUMMARIES AND CONCLUSIONS

Summary of Study Findings

Study #1 Summary

Respiratory load compensation is a sensory-motor reflex generated in the brainstem

respiratory neural network. The NTS is thought to be the primary structure to process

the respiratory load related afferent activity and contribute to the modification of

breathing pattern by sending efferent projections to other structures in the brainstem

respiratory neural network, such as the VRC and PRG. The sensory pathway and motor

responses of respiratory load compensation have been extensively studied; however,

the mechanism of neurogenesis of load compensation is still unknown. A variety of

studies have shown that inhibitory interconnections between the brainstem respiratory

groups play critical roles for the genesis of respiratory rhythm and pattern. The purpose

of this study was to examine whether the inhibitory glycinergic neurons in the NTS were

activated by ITTO in anesthetized animals. The results showed that load compensation

responses, including prolonged Ti, Te and Ttot were elicited by ITTO. In addition,

glycinergic neurons in both rNTS and cNTS were activated in anesthetized animals with

ITTO compared with the sham group. The results suggest that these activated inhibitory

glycinergic neurons in the NTS might be essential for the neurogenesis of load

compensation responses in anesthetized animals.

Study #2 Summary

This study was performed to test whether ETTO would elicit activation of inhibitory

glycinergic neurons in the NTS as found in the first study (Chapter 3). In addition, we

investigated the effect of vagotomy on the respiratory load compensation responses

111

and glycinergic neuron expression in the NTS. The results showed that ETTO produced

load compensation responses with increased Ti, Te, Ttot as well as elevated activation

of inhibitory glycinergic neurons in the NTS. Vagotomized animals receiving ETTO did

not exhibit these load compensation responses. In addition, vagotomy significantly

reduced the activation of inhibitory glycinergic neurons in the cNTS and rNTS

Study #3 Summary

Conscious animals respond to respiratory loads differently than anesthetized animals

due to cortical and subcortical processing. Conscious animals behaviorally control

breathing pattern in response to respiratory challenges. Therefore, we hypothesized

that the mechanism of neurogenesis of load compensation in the brainstem in

conscious animals would differ from anesthetized animals. In the present study, we

examined the load compensation responses in conscious animals with sustained

respiratory loads. In addition, we determined if inhibitory glycinergic neurons would be

activated in the NTS in conscious animals as we found in anesthetized animals. The

results demonstrated that conscious animals responded to ITTO with prolongation of Te

and Ttot not Ti which is different from anesthetized animals. Moreover, glycinergic

neurons were activated in the rNTS but not in the cNTS. These results suggest that the

activated glycinergic neurons in the rNTS are more important for the neurogenesis of

load compensation responses than cNTS in conscious animals.

Study #4 Summary

Emotional state can modulate the perception of respiratory loads but the range of

respiratory load magnitudes affected by emotional state is unknown. We hypothesized

that viewing pleasant, neutral and unpleasant affective pictures would modulate the

perception of respiratory loads of different load magnitudes. Twenty-four healthy adults

112

participated in the study. Five inspiratory resistive loads of increasing magnitude (5, 10,

15, 20, 45 cmH2O/L/sec) were repeatedly presented for one inspiration while

participants viewed pleasant, neutral and unpleasant affective picture series.

Participants rated how difficult it was to breathe against the load immediately after each

presentation. Only at the lowest load, magnitude estimation ratings were greater when

subjects viewed the unpleasant series compared to the neutral and pleasant series.

These results suggest that negative emotional state increases the sense of respiratory

effort for single presentations of a low magnitude resistive load but high magnitude

loads are not further modulated by emotional state.

Respiratory Load Compensation in Anesthetized and Conscious Animals

Individuals are able to compensate for respiratory challenges by modifying their

breathing pattern for the maintenance of homeostasis that is called respiratory load

compensation. Load compensation responses are characterized by a prolongation of

breath duration, a decrease in tidal volume and recruitment of respiratory muscle

activity (Clark and von Euler, 1972; Koehler and Bishop, 1979; Lopata et al., 1983;

Zechman et al., 1976). In study 1 (Chpater 2), ITTO let to significantly prolongation of Ti,

Te and Ttot, consistant with previous studies (Bernhardt et al., 2011a; Pate and

Davenport, 2012b). During the O phase, Ti was significantly increased only during O1,

whereas Te was prolonged during the entire occluded period. This may be due to the

ITTO initiated during the expiratory phase of breathing. We attempted to initiate ITTO at

FRC; however, the breathing frequency in rats is 85-110 breaths/min which is too fast to

initiate ITTO at specific breath phases with accurate timing, especially during inspiration

as it is shorter than expiration. In addition, acute ITTO did not enhance inspiratory

muscle outputs in the present study, whereas chronic ITTO conditioning has been

113

shown to be associated with type II fiber hypertrophy in the medial costal region of the

diaphragm (Smith et al., 2012). This indicates that acute ITTO is a good model to study

respiratory load compensation responses and chronic ITTO might be a good animal

model for clinical respiratory muscle strength training, including individuals with stroke

or spinal cord injury, or weaning patients from mechanical ventilators.

In conscious animals, repetitive application of acute ITTO might cause aversive

respiratory sensations. We have observed that conscious animals will hold their

breathing during expiration instead of breathing through the occlusion loads presumably

to alleviate the aversive sensation which is a fear escape response. Previous studies

have demonstrated that chronic ITTO conditioning changed gene expression in the

medial thalamus, an integral region involved in the processing of respiratory sensory

pathway (Bernhardt et al., 2011b) and ITTO conditioning caused stress, anxiety,

depression and neural state changes (Pate and Davenport, 2012a). In the present study,

conscious rats showed the fear escape response in the face of acute ITTO which may

ultimately evolve into depression and anxiety. In addition, studies have shown that

patients with respiratory obstructive diseases often present with affective disorders,

such as anxiety and/or panic (Brenes, 2003; Wagena et al., 2005). However, the

reasons for the expression of affective disorders in respiratory obstructed patients are

still not clear. Based on the present and previous studies, we suggest that animals that

experience repeated respiratory obstruction will behaviorally modify their breathing

pattern to avoid aversive respiratory sensations and eventually develop affective

disorders due to chronic respiratory obstructed breathing.

114

Effect of Emotion on Respiratory Perception

Acute ITTO in conscious rats elicited behavioral fear escape response (Chapter

4) and chronic ITTO conditioning caused anxiety and panic (Pate and Davenport,

2012a). Studies have shown that negative affect (NA) affected the accuracy of

respiratory perception in patients with respiratory obstructive diseases regardless of

their respiratory function. For example, COPD or asthmatic patients with higher NA

reported more intense symptoms compared to patients with lower NA (De Peuter et al.,

2008; Vogele and von Leupoldt, 2008). Over-perception of respiratory loading may

cause overuse of medications or activity avoidance (Main et al., 2003; Ng et al., 2007;

von Leupoldt and Dahme, 2007). Therefore, it is important to provide respiratory

obstructed patients with high NA appropriate and timely treatments. Although emotional

states affect the perception of respiratory loads, it is still unknown whether emotional

modulation of respiratory perception is different at varying levels of respiratory

restriction. The results of study 4 (Chapter 5) demonstrated that picture elicited NA state

increased the perceived respiratory effort for low magnitude resistive loads but not for

high magnitude loads. This suggests that respiratory perception in patients with mild

COPD or asthma may have less modulation by NA state than patients with severe

COPD or asthma since the physiological discomfort overrides the emotional impact.

Activation of Inhibitory Glycinergic Neurons in the NTS

In anesthetized animals, ITTO activated inhibitory glycinergic neurons in both of

the caudal and rostral NTS. In conscious animals, inhibitory glycinergic neurons were

only activated in the rNTS. The differences in the results indicate that load

compensation responses may be processing differently in anesthetized and conscious

animals.

115

In anesthetized animals, ITTO- or ETTO-activated glycinergic neurons were

concentrated in the SolM and SolV subdivisions of the NTS. In conscious animals,

glycinergic neurons were activated by ITTO in the SolM, SolV and SolVL.

The different subdivisions of the NTS play different roles in the control of breathing.

It has been demonstrated that interneurons in the SoM are associated with the Hering-

Breuer reflex. Excitation of interneurons in the SolM resulted in termination of inspiration

and prolongation of expiration that was transiently impaired by blockade of excitatory

amino acid neurotransmitters (Bonham and McCrimmon, 1990). In contrast, deletion of

SolVL did not impair this respiratory reflex (McCrimmon and Alheid, 2004). In addition,

interneurons in the SolM send projections to the rostral VRC, including BötC and Pre-

BötC, but interneurons in the SolVL project broadly to the VRC (Alheid et al., 2011). It

has been demonstrated that interneurons in the SolVL receive projections from cortical

and subcortical structures (Danielsen et al., 1989; van der Kooy et al., 1984). Therefore,

we proposed that these activated inhibitory glycinergic neurons in the SolM and SolV

may be the p-cells responsible for the reflex load compensation responses. The

activated glycinergic neurons in the SolVL may be activated by higher cortical and

subcortical structures for the behavioral control of breathing pattern.

Significance and Application of the Study

COPD and asthma are the two main respiratory obstructive diseases

characterized by airflow limitation. The airflow limitation is mostly the result of an

increased airway resistance (airway obstruction) acting as a mechanical impedance to

airflow (Hogg, 2004; Saetta and Turato, 2001; Turato et al., 2001). Before airflow

limitation of patients with COPD and asthma reaches a critical level, their central

respiratory control neural mechanism will adapt to the increased airway resistance by

116

changing breathing patterns to sustain alveolar ventilation, which is respiratory load

compensation. Respiratory load compensation is a sensory-motor reflex resulting from a

reconfiguration of the brainstem respiratory neural network. Reconfiguration of the

neural network is a consequence of dynamic changes of synaptic interconnections

between respiratory-related neurons in the brainstem. A better understanding of the role

of the central neural mechanism responsible for respiratory load compensation

undoubtedly provides an opportunity to develop novel therapeutic approaches for these

respiratory diseases in these patients.

ITTO is a good strategy to simulate patients with respiratory obstructive diseases.

ITTO successfully elicited load compensation responses and activated a variety of

neural structures in the brainstem including the NTS, periaqueductal gray and nucleus

ambiguus in anesthetized animals (Pate and Davenport, 2012b). In study 1 (Chapter 2)

and 2 (Chapter 3), it is further demonstrated that these ITTO- or ETTO activated

interneurons in the NTS that are glycinergic. These glycinergic neurons are consistent

with the role of inhibitory p-cells in the Rybak et al. model for generating the load

compensation responses (Rybak et al., 2007; Rybak et al., 2008). Study 3 (Chapter 4)

and 4 (Chapter 5) demonstrated that glycinergic neurons in the NTS play a reduced role

for the neurogenesis of load compensation responses in conscious animals. In addition,

affective states would change perceived respiratory effort and add behavioral

modulation to the load compensation responses. These findings advance and expand

our knowledge for the modeling of the respiratory neural network in the brainstem, as

well as cortical and subcortical regions.

117

Direction for Future Studies

Future studies are envisioned to combine a multi-electrode array recording

systems and computational methods to investigate the cross-correlation between

neurons in the NTS and VRC during respiratory mechanical loads. We aim to

demonstrate the presence of an inhibitory projection from the NTS to the VRC.

Conclusions

This dissertation examined an inhibitory neurotransmitter released by NTS

interneurons activated by respiratory loads in anesthetized and conscious animals as

well the effect of emotion on the perceived respiratory effort in humans. The activated

interneurons in the NTS were found to be inhibitory glycinergic in both anesthetized and

conscious animals. These inhibitory glycinergic neurons may play a more important in

anesthetized animals compared with conscious animals that will also behaviorally

modify breathing pattern. In addition, affective states affected perceived respiratory

effort in humans with negative affective state resulting in greater perceived respiratory

effort compared with neutral and positive affective states.

118

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BIOGRAPHICAL SKETCH

Hsiu-Wen Tsai was born in Hsinchu, Taiwan. She received her Bachelor of Science

degree in nutrition and human sciences in Taipei Medical University in Taipei, Taiwan in

2002. She received her Master of Science degree in biochemistry and molecular biology

in National Chang-Kung University in Tainan, Taiwan in 2004. She joined the laboratory

of Dr. Paul W. Davenport in Department of Physiological Sciences, College of

Veterinary Medicine in University of Florida to study respiratory physiology and

neurophysiology since August 2008. Her research interests include neurotransmission,

respiratory sensation and respiratory mechanism.

tracheal occlusion evoked respiratory load ...special thanks go to my parents, mr. chung-shun tsai...

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