TRACHEAL OCCLUSION EVOKED RESPIRATORY LOAD ...Special thanks go to my parents, Mr. Chung-Shun Tsai...
Transcript of 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
Breathing Pattern ............................................................................................. 39
Immunofluorescence Double-Staining .............................................................. 40
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
Materials and Methods ............................................................................................ 53
Animals ............................................................................................................. 53
Surgical Procedure ........................................................................................... 53
Experimental Protocol ...................................................................................... 55
Immunofluorescence Double-Staining Protocol ................................................ 55
Data Analysis ................................................................................................... 56
Statistical Analysis ............................................................................................ 56
Results .................................................................................................................... 56
Breathing Pattern ............................................................................................. 56
Immunofluorescence Double-Staining .............................................................. 58
Discussion .............................................................................................................. 59
Breathing Pattern ............................................................................................. 59
Immunofluorescence Double-Staining .............................................................. 60
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
Materials and Methods ............................................................................................ 79
Animals ............................................................................................................. 79
Surgical Procedure ........................................................................................... 79
Experimental Protocol ...................................................................................... 80
Immunofluorescence Double-Staining Protocol ................................................ 81
Data Analysis ................................................................................................... 81
Statistical Analysis ............................................................................................ 81
Results .................................................................................................................... 81
Breathing Pattern ............................................................................................. 81
lmmunofluorescence Double-Staining .............................................................. 81
Discussion .............................................................................................................. 82
Breathing Pattern ............................................................................................. 82
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Immunofluorescence Double-Staining .............................................................. 83
5 THE IMPACT OF EMOTION ON THE PERCEPTION OF GRADED MAGNITUDES OF RESPIRATORY RESISTIVE LOADS ....................................... 95
Background ............................................................................................................. 95
Materials and Methods ............................................................................................ 97
Participants ....................................................................................................... 97
Affective Picture Series .................................................................................... 97
Inspiratory Resistive Loads .............................................................................. 97
Measurement of Respiration ............................................................................ 98
Measurement of Emotional Responses ............................................................ 98
Procedure ......................................................................................................... 98
Statistical Analysis ............................................................................................ 99
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
22
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.
Immunofluorescence Double-Staining
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.
Materials and Methods
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
experimental protocol was reviewed and approved by the Institutional Animal Care and
Use Committee of the University of Florida.
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
Experimental Protocol
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.
Immunofluorescence Double-Staining Protocol
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
Immunofluorescence Double-Staining
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).
Immunofluorescence Double-Staining
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
O1 vs. R3 N/A N/A N/A <.01 N/A
O2 vs. R1 N/A N/A N/A <.001 N/A
O2 vs. R2 N/A N/A N/A <.01 N/A
O2 vs. R3 N/A N/A N/A <.01 N/A
O3 vs. R1 N/A N/A N/A <.001 N/A
O3 vs. R2 N/A N/A N/A <.01 N/A
O3 vs. R3 N/A N/A N/A <.01 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.
Materials and Methods
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
experimental protocol was reviewed and approved by the Institutional Animal Care and
Use Committee of the University of Florida.
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.
Experimental Protocol
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.
Immunofluorescence Double-Staining Protocol
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.
Statistical Analysis
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.
Immunofluorescence Double-Staining
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).
Materials and Methods
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.
Statistical Analysis
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
Pleasant Neutral Unpleasant
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
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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
Pleasant Neutral Unpleasant
Slo
pe o
f Lo
gM
E-L
ogP
ma
x
0.0
0.5
1.0
1.5
2.0
2.5
*
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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
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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.