Citation
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

Material Information

Title:
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
Creator:
Tsai, Hsiu-Wen
Publisher:
University of Florida
Publication Date:
Language:
English

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Veterinary Medical Sciences
Veterinary Medicine
Committee Chair:
Davenport, Paul W
Committee Members:
Hayward, Linda F
Fuller, David
Reep, Roger L
Bolser, Donald Clementz
Sapienza, Christine M
Graduation Date:
5/4/2013

Subjects

Subjects / Keywords:
Brain stem ( jstor )
Breathing ( jstor )
Expiration ( jstor )
Fluorescent antibody techniques ( jstor )
Inspiration ( jstor )
Lungs ( jstor )
Neurons ( jstor )
Neurotransmitters ( jstor )
Rats ( jstor )
Respiratory mechanics ( jstor )
glycine
respiration
City of Davenport ( local )

Notes

General Note:
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 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.

Record Information

Source Institution:
UFRGP
Rights Management:
Copyright Tsai, Hsiu-Wen. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Embargo Date:
5/31/2015

Downloads

This item has the following downloads:


Full Text

PAGE 1

1 TRACHEAL OCCLUSION EVOKED RESPIRATORY LOAD COMPENSATION AND INHIBITORY NEUROTRANSMITTER EXPRESSION IN THE NUCLEUS OF SOLITARY TRACT IN ANIMALS AND THE EFFECT OF EMOTI ON ON THE PERCEPTION OF RESPIRA T O RY 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 201 3

PAGE 2

2 201 3 Hsiu Wen Tsai

PAGE 3

3 To my parents and my husband

PAGE 4

4 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 f ocus in teaching and mentoring. I was privileged to have a dissertation committee matching my research interests. M y sincere gratitude is given to my supervisory committee members : Dr. Don ald Bolser, Dr. David Fuller, Dr Linda Hayward Dr. Roger Reep and Dr. Christine Sapienza for their helpful suggestions and advice s that made the dissertation more meaningful and complete. I am grateful to have the o pportunity 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 t hank Dr. Andreas von Leupoldt for his help ful suggestions and insights into psychology. I am grateful to Dr. Pei Ying Sarah Chan for her help support and friendship I also t hank Dr. Kun Ze Lee for his help and encourageme nt 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 M r s. Guei Hua T ai 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 co uld 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

PAGE 5

5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 TABLE OF CONTENTS ................................ ................................ ................................ .. 5 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURE S ................................ ................................ ................................ .......... 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. Id entification of I nhibitory N eurotransmitters in NTS N eurons A ctivated by ETTO in A nesthetized R ats with V agi I ntact and V agotomized. ................................ ................................ ................................ 26 Specific Aim 3. Identification of the L oad C ompensation R esponses and the I nhibitory N eurotransmitters in the ITTO A ctivated N eurons in the NTS of C onscious R ats. ................................ ................................ ............................ 27 Specific Aim 4. Identification of the E ffect of D ifferent E motional S tates on the P erception of G raded R espiratory R esistive L oads in C onscious H umans. ................................ ................................ ................................ ........ 28 2 THE EFFECT OF TRACHEAL OCCLUSIONS ON RESPIRATORY LOAD COMPENSATION AND CHANGES IN NEURONS CONTAINING INHIBITORY NEUROTRANSMITTER IN THE NUCLEUS OF THE SOLIT ARY TRACT IN ANESTHETIZED RATS ................................ ................................ .......................... 30 Background ................................ ................................ ................................ ............. 30 Materials and Methods ................................ ................................ ............................ 32 Animals ................................ ................................ ................................ ............. 32 Surgical P rocess ................................ ................................ .............................. 32 Experimental Protocol ................................ ................................ ...................... 34 Immunofluorescence Double Staining Protocol ................................ ................ 34 Data A nalysis ................................ ................................ ................................ ... 35 Breathing pattern ................................ ................................ ....................... 35

PAGE 6

6 Immunofluo rescence double staining ................................ ........................ 36 Statistical analysis ................................ ................................ ...................... 37 Results ................................ ................................ ................................ .................... 37 Breathing Pattern ................................ ................................ ............................. 37 Immunofluo rescence D ouble S taining ................................ .............................. 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 P roce dure ................................ ................................ ........................... 53 Experimental Protocol ................................ ................................ ...................... 55 Immunofluorescence D ouble S taining P rotocol ................................ ................ 55 Data A nalysis ................................ ................................ ................................ ... 56 Statistical A n alysis ................................ ................................ ............................ 56 Results ................................ ................................ ................................ .................... 56 Breathing P attern ................................ ................................ ............................. 56 Immunofluorescence D ouble S taining ................................ .............................. 58 Discussion ................................ ................................ ................................ .............. 59 Breathing P attern ................................ ................................ ............................. 59 Immunofluorescence D ouble S taining ................................ .............................. 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 P rocedure ................................ ................................ ........................... 79 Experimental P rotocol ................................ ................................ ...................... 80 Immunofluorescence D ouble S taining P rotocol ................................ ................ 81 Data Analysis ................................ ................................ ................................ ... 81 Statistical A n alysis ................................ ................................ ............................ 81 Results ................................ ................................ ................................ .................... 81 Breathing P attern ................................ ................................ ............................. 81 lmmunofluo rescence Double Staining ................................ .............................. 81 Discussio n ................................ ................................ ................................ .............. 82 Breathing P attern ................................ ................................ ............................. 82

PAGE 7

7 Immunofluorescence D ouble S taining ................................ .............................. 83 5 THE IMPACT OF EMOTION ON THE PERCEPTION OF GRADED MAGNITUDES OF RESPIRATORY RESISTIVE LOADS ................................ ....... 95 Background ................................ ................................ ................................ ............. 95 Materials and Methods ................................ ................................ ............................ 97 Partic ipants ................................ ................................ ................................ ....... 97 Affective P icture S eries ................................ ................................ .................... 97 Inspiratory R esistive L oads ................................ ................................ .............. 97 Measurement of R espiration ................................ ................................ ............ 98 Measurement of E motional R esponses ................................ ............................ 98 Procedure ................................ ................................ ................................ ......... 98 Statistical A nalysis ................................ ................................ ............................ 99 Results ................................ ................................ ................................ .................... 99 Discussion ................................ ................................ ................................ ............ 101 6 SUMMARIES AND CONCLUSIONS ................................ ................................ .... 110 Summary of Study Findings ................................ ................................ .................. 110 St udy #1 Summary ................................ ................................ ......................... 110 Study #2 Summary ................................ ................................ ......................... 110 Study #3 Summary ................................ ................................ ......................... 111 Study #4 Summary ................................ ................................ ......................... 111 Respirator y 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

PAGE 8

8 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

PAGE 9

9 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 lo ad compensation responses in anesthetized animals. ......... 45 2 3 ITTO effect on EMGdia activity and Pes in anesthetized animals. ..................... 46 2 4 ITTO activated inhibitory gly cinergic 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 r NTS in anesthetized animals. ................................ ................................ ......................... 50 2 8 Immunofluorescence double staining of c Fos and GlyT2 in the combined rNTS and c NTS in anesthetized animals. ................................ ........................... 51 3 1 Effect of vagotomy on the respiratory load compensation response s ................ 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. ................................ 6 8 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

PAGE 10

10 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 activati on 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 c NTS in conscious animals. ................................ ............................... 94 5 1 Schematic of exp erimental 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

PAGE 11

11 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 TRACH EAL OCCLUSION EVOKED RESPIRATORY LOAD COMPENSATION AND INHIBITORY NEUROTRANSMITTER EXPRESSION IN THE NUCLEUS OF SOLITARY TRACT IN ANIMALS AND THE EFFECT OF EMOTI ON ON THE PERCEPTION OF RESPIRA T O RY LOAD IN HUMANS By Hsiu Wen Tsai May 201 3 Chair: Paul W. Davenport Major: Veterinary Medical Sciences An imals are able to adapt to respiratory mechanical load by c hanging their breathing pattern that is defined as r espiratory load compensation. Load compensation is mainly dominated by r eciprocal inhibitory inter connections between brainstem respiratory related neurons in anes thetized animals but involves cortical and subcortical processing in consci ous animals. The n ucleus of the solitary tract (NTS) is the principal site that receives and integrates respira tory peripheral afferents for the neurogenesis of load compensatio n. The central aim of this study is to investigate neurons that release inhibitory neurotransmitters in the NTS that may be critical ly 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 perc eption in conscious humans. The f irst study showed intrinsic, tran sient t racheal occlusions (ITTO ) elicited load compensation as evidenced by prolong ed 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

PAGE 12

12 external, transient tracheal occlusion s (ETTO ) also elicited load compensation with prol ongation of Ti, Te and Ttot as well as activated glycinergic neuro ns in the NTS of anesthetized animals The load compensation responses were abolished by bilateral cervical vagotomy and the activa tion of glycinergic neurons was suppressed in the caudal NTS (cNTS) and rostral NTS (rNTS) T he third study demonstrated in conscious animals ITTO elicited behavioral control of load compensation with prolongation of only Te and Ttot at the third occlude d breath for each series of ITTO I nhibitory g lycinergic neurons wer e only activated in the r NTS 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 effo rt for a single presentation o f low magnitude resistive load but high magnitude loads were not m odulated by emotional states. The resu lts of these studies suggest th at inhibitory g lycinergic neurons in the NTS play a significant role in the vagally mediated neurogenesis of load compensat ion in anesthetized animals that is greater than conscious animals. In conscious animals and humans, cortical and subcortical processing is i nvolve d in the r espiratory loa d compensation responses.

PAGE 13

13 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 challeng es, animals are able to compensate to the stimuli by adjusting tidal volume (V t ) and /or breathing frequency (fb) to maintain appropriate minute ventilation A fferent input s and motor output s of the respiratory neural network during eupneic breathing and respiratory load compensation have been extensively studied in anestheti zed animals T he neurotransmitters within the neurons of the respiratory neural network responsible for the neurogeneis of breathing have been studied However, the neurons activated during respira tory load compensation response s and the neurotransmitters within those neurons are unknown. I t 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 characteri zed as the respiratory vo lume timing (V t T) relationship in anesthetized animal s by Clark and von Euler ( Clark and von Euler, 1 972 ) They demonstrated that Ti was inversely r elated to the Vt and Te was d ependent on Ti of the same breath. T he Vt T relationship was abolished by vago tomy in anes thetized ca ts, indicating that the vagi are the principal respiratory afferents transducing information related to the change of lung volume to t he respiratory neural network to elicit the Vt T response The V t T relationship has also been observed in consciou s human s but only at a Vt at least two times of eupn eic values ( Clark and von Euler, 1972 ) Subsequently Zechman an d colleagues ( Zechman et al., 1976 ) used an

PAGE 14

14 e xtrinsic respiratory resistive loading model to study the re spiratory reflex control system in response to respiratory mechanical stimuli They demonstrated that applying a respiratory resistive load either to inspiration or expiration increased airway res istance which in turn decreas ed 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 unchan ged. However, applying a single e xpiratory resistive load decreased inspiratory duration due to an upward shift of fu nctional residual capacity T he results imply tha t the V t T relationship is control led differently during inspira tion and expiration Davenport et al. ( Davenport et al., 1981a ; Davenport et al., 1984 ) demonstrated that s lowly adapting pulmonary stretch receptors ( PSR s ) located in the s mooth muscle of the airways innervated by fast conducting, myelinated aff erent fibers in the vagus nerve s respond to changes in either lung volume and smooth muscle tone and mediate respira t o ry load compensation response s PSR s are activated differently during inspiration and expiration. During inspiration, lung inflation elevates PSR activity by increasing the frequency of PSR discharge. During expi ration, lung deflation activate s PSR by increasing the spike number ( Davenport and Wozniak, 1986 ) Vagally mediated PSR a fferents principally terminate o n the second order inter neurons 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 pr incipal structure to process peripheral respiratory afferents. It is impo rtant to study the neurotransmitters released by interneurons in t he NTS for further understand the neurogenesis of respiratory load compensation responses.

PAGE 15

15 Central Control of Breathing Autonomic r espiration is a f eedback control system composed of three elements: brainstem central controller, effectors (respiratory muscles) and s ensors (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 b rainstem can be characterized on t he basis of their dis charge 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) an d 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 cl assified into various subcategories a ccording to 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 e xhibit a peak discharge in early inspiration and decline gradually throu ghout 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 a n 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 begi n to fire in e xpiration increase their discharge frequency and reach peak discharge during the late expirat ory phase Late I neurons only discharge in late inspiration and early

PAGE 16

16 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 responsibl e for terminating expiration and initiating the onset of the next inspiration. EI and I E 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 respir atory phase transition. Pontine inspira t ory, expiratory and IE neu rons 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 output s from the VRC to the pons during eupnea ( Rybak et al., 2008 ) The D RG is mainly composed of inspiratory related bulbospinal and propriobulbar neuron s The distribution of projections from the DRG is variable in different species. In cat s 50 90% of the inspiratory neurons (ramp I, early I ) send projections to the phrenic and external intercostal motor neurons in the spinal cord ( Berger, 1977 ; Cohen et al., 1974 ; Lipski et al., 1983 ) ; whereas i n rats, less than 20% of D RG neurons project to the motorn eurons in spinal cord ( de Castro et al., 1994 ) The VRC is located i n the vetrolateral medulla and contains several nuclei including the Btzinger complex (BtC), the pre Btzinger complex (pre BtC), the rostral (rVRG) and caudal (cVRG) parts of the VRG. The BtC neurons are the princip al source of expiratory inhibition dur ing eup n e ic breathing P ost I and aug E neurons in the BtC are mutually inhibited and make widely distributed inhibitory interconnections with other respiratory components in the pons and medulla ( Ezure and Manabe, 1988 ; Ezure et

PAGE 17

17 al., 2003b ; Jiang and Lipski, 1990 ; Merrill et al., 1983 ; S aito et al., 2002 ; Tian et al., 1999 ) Post I and aug E neurons in the BtC inhibit pre I and early I neurons in the pre BtC to inhibit inspiratory output s during expiration. Pre I neurons in the pre BtC are less inhibited by the post I neurons in the BtC during the late expiration to participate in the transition from expiration to inspiration. Pre I neurons in the pre BtC have intrinsic persistent sodium current dependent (I Nap ) rh ythm o gen ic propert ies that are inactivated by tonic excitatory input s and phasic inhibitio n from post I and aug E neurons in the BtC during normal breathing but operate phasically to generate intrinsic rhyth mic 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 BtC neurons activate ramp I neurons in the r VRG and hypoglossal motor neurons to initiate inspirat ion ( Feldman and D el 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 BtC provide inhibitory projecti ons to other com ponents in the network to coo rdinate breathing pattern ( Ezure, 1990 ; Sun et al., 1998 ) The VRG contains main ly bilateral clusters of bulbosp inal respiratory neurons and is subdivided into rVRG and cVRG R amp I neurons are pre dominantly 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 output s ( Ezure, 1990 ) The respiratory control system is regulated by feedback s from chemoreceptors and mechanoreceptors C entral chemoreceptors in the retrotrapezoid nucleus (RTN) and

PAGE 18

18 peripheral chemoreceptors in the aortic and ca rotid bodies sense the changes in the concentration of oxygen and carbon dioxide in the cerebrospinal fluid and blood M echanoreceptors in the lung s and airway s including PSR s rapidly adapting receptors (RARs) and C fibers monitor the changes in the airflow and the volume of air in the lung s Mechanoreceptors in the respiratory muscles such as muscle spindles, G olgi tendon organ s and joint receptors sense the changes in the tens ion 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 nerve s to connect with the respiratory central control neural network ( Kubin et al., 2006 ) PSRs in the smooth muscles of the airw ays are activated by lung inflation and terminate on second order interne urons (pump cells: p cells; inspiratory in the i pisilateral intermediate, interstitial, ventral and ventro lateral 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 commi ssural and intermediate sub nuclei of the NTS ( Kubin et al., 2006 ) Bronchopulmonary C fibers are activated by external irritants and term inate in the parvicellular nucleus, medial and commissural nucleus of the NTS a nd the area postrem a ( Kubin et al., 2006 ) The se respiratory sensory inputs are mainly terminated and processed in different subnuclei of the NTS The second order NTS inter neurons in turn project widely throughout the PRG and VRC to shape and modulate inspira tory and expiratory motor outputs as well as to produce normal breathing pattern ( Ezure et al., 2002 )

PAGE 19

19 Respiratory muscles are composed of laryngeal and pump skeletal muscles. Respiratory laryngeal muscles in the upper airways regulate the airway resistance during inspiration and exp iration The d iaphragm, intercostals and abdominal muscles are respiratory pump muscles. The d iaphragm and external inter costals are the major inspiratory muscles innervated by phrenic and intercostal motorneurons in the spinal cord, respectively. The internal intercostals and abdominal muscles are the princip al expiratory muscles innervated by thoracic and lumbar motorneuro ns respectively. The movement s of the respiratory laryngeal and pump mus cles 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 modifi ed 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 sys tem in the thalamus which determine whether respiratory afferents will or will not r each the higher cortical processing area s ( Chan and Davenport, 2008 ; Davenport and Vovk, 2009 ) An a ppropriate r espirator y gate system is considered to be a protective mechanism for homeostasis that helps animals to be aware and behaviorally adjust their breathing pattern in respon se to respiratory challenges. Respiratory afferents that are gated into the cortical and subcortical regions mainly project to the prefrontal c ortex, limbic system and

PAGE 20

20 soma t osensory cortex Somatosensory areas integrate these signal s and send projection s to the motor cortex. The m otor cortex control s the respiratory muscles through the cortico s pinal pathway to generate voluntary control of breathing ( Davenport and Vovk, 2009 ) Neurotransmitters i n the Control of Breathing Neurons in th e respiratory neural control network utilize neu rotrans mitters as mediators for signal transmission to generate normal rhythmic breathing pattern s It is of critical importance to identif y the neurotransmitters which a neuron releases to define the role it play s with in 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 measurin g the excitatory (EPSPs) or inhibitory (I P SPs ) post synaptic potentials elic ited from an identified neuron respectively Imm u nohistochemist r y staining is capable of identifying the morpholog y of a neuron including its den d ritic fields, axonal projections and the neurotrans mi t ters it secret e s. A variety of studies combined these two methods to identify the chemical neuroanatomy of breathing in the brainstem ( Alheid and McCrimmon, 2008 ; Stornetta, 2008 ) Ther e 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 a nd motor neurons ( Bonham, 1995 ; Haxhiu et al., 2005 ) Glutamate mainly acts at non N methyl D aspartate (non NMDA) receptors such as amino 3 hydroxy 5 m ethylisoxazole 4 propionic acid (AMPA) and kainate within the networ k. Most of the

PAGE 21

21 cVRG bulbospinal neurons with a firing pattern that increase d 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 an d Feldman, 1984 ; Cohen et al., 1974 ) In addition, peripheral chemoreceptors relayed to second order inter neurons in the commissural sub nucleus of the NTS are mainly excitatory ( Finley and Katz, 1992 ; Otake et al., 1993 ) Inhibitory neurotransmitters play a critical role in controlli ng neuronal excitability in the respiratory neural network GABA mainly acts on GABA A receptors and glycine binds to glycine receptors that are chloride ion channel s ( Jentsch et al., 2002 ) Fa s t 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 den sely distributed in the rostral brains tem 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 o f medullar y 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 inspiratio n and post I neurons receive IPSP s during late expiration ( Ballantyne and Richter, 1984 1986 ) I t is

PAGE 22

22 apparent that GABA and g lyc ine mediated postsynaptic inhibitory inputs not only prevent spiking of neurons dur ing silent periods but also act in concert with excitatory synaptic inputs during the active periods to help shape the pattern of the respiratory motor output s ( 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 ; E zure 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 depend ent synaptic inhibition with glycine and GABA A receptor antagonists sto ps respiratory rhythmogenesis in mature animals ( Paton et al., 1994 ; Paton and Richter, 1995 ) Local application of glycine by mi croinjection to the respiratory related area in the brainstem decreases the firing rate of bulbar respira t ory neurons ( Bianchi et al., 1995 ) Application of the glycine antagonist strychnine depolarizes respiratory neu rons in all phases of respiration indicating that these neurons are tonically inhibited during breathing ( Bianchi et al., 1995 ) In the brainste m respiratory network, a variety of respiratory neuron s secret e glycine. A ug E neurons in the BtC are glycinergic ( Schreihofer et al., 1999 ) Post I neurons in the ventrolateral medulla secret e glycine ( Ezure et al., 2003a ) In addition, the PSR activated second order interneurons in the ventrolateral interstitial subnucleus of t he NTS appear t o be inhibitory and mainly secret e GABA ( Takakura et al., 2007 ) but also co release glycine (26%) ( Kubin et al., 2006 ) Therefore, in the respiratory control

PAGE 23

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 o blongata is subdivided into several regions based on cytoarchitect onics, including the commis s ua l ( SolC ) dorsal (SolD), dorsal lateral (SolDL), gelatinosus (SolG), interstitial ( SolI ) intermediate (SolIM), medial (SolM), ventral (SolV) and ventolateral (SolVL) ( Kalia a nd Mesulam, 1980a b ; Kalia and Sullivan, 1982 ) The NTS is the princip al site that receives and integrate s 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 ner vous system for homeostasis and autonomic regulation Peripheral afferents from different visceral systems t erminate in different subdivisions o f the NTS. Afferents from the respiratory, cardiovascular and gastrointe stinal systems mainly terminate in the caudal two t hirds 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 dis charging patterns have also bee n 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 Sol VL rostral to the obex. Inspira t ory neurons

PAGE 24

24 are found in the Sol VL 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 t he princip al site of the first synapse in afferent elicited respiratory reflex es and might play an important role in respiratory pattern configuration S timulation of vagal afferents and lung inflation are demonstrated to activate SARs in the lungs and airways that terminate on the second order inter neurons 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 BtC and rVRG to inhibit inspiration. ( Kubin et al., 2006 ; Rybak et al., 2007 ; Rybak et al., 2008 ; Subramanian et al., 2007 ) Base d on results from immunohistochemistry, most peripheral afferents projecting to the NTS utilize glutamate as an excitatory ne uro r t ansmitter ( Sykes et al., 1997 ) The inhibitory neuro transmitter g lycine is frequently distributed in the SolI, SolDL and SolVL subnuclei of the NTS ( Tanaka et al., 2003 ) The other inh ibitory 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, i t has been demonstrated that there are more GABAergic neuro ns in the rostral brainstem, whereas glycinergic neurons are m ore densely located in the caudal brainstem and spinal cord ( Araki et al., 19 88 ; Nicoll et al., 1990 ; Elekes et al., 1986 ; Lloyd et al., 1983 ) This implies that th e inhibitory

PAGE 25

25 neurotransmitters might play a critical role in the NTS for the integration of respiratory afferents f or the modification of breathing patterns. The purpose of th is study is to examine t he distribution of neurons that release inhibitor y ne u rotran s mitter in the NTS in response to respiratory resistive load ing in both anesthetized and conscious animals. Specific Aims Spe cific Aim 1. Identification of I nhibitory N eurotransmitters i n ITTO A ctivated N eurons in the NTS of A nesthetized R ats Rationale: ITTO is an animal model that ha s 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 th e 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 onl y shows that a neuron was activ ated but it is not able to distinguish if the neuron uses excitatory or inhibitory neurotransmitters. The NTS is the primary region that i ntegrates 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. Activatio n 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 inhibit ion of inspiration and prolongation of expiratory duration. The b rainstem respiratory neural network is mainly constructed by

PAGE 26

26 inhibitory reciprocal interconnections rather than excitatory synapses ( Rybak et al., 2007 ; Rybak et al., 2008 ; Smith et al., 2007 ) Theref ore, we hypothesized that ITTO will elicit a load compe nsation modulation of breathing pattern and ITTO activated neurons in the NTS will be inhibi tory glycinergic neurons in anesthetized animals. H ypothesis: Acute ITTO activated neurons in the NTS of anesthetized rats will be inhibitor y glycinergic neurons. Spe cific Aim 2. Identification of I nhibitory N eurotransmitters in NTS N eurons A ctivated by ETTO in A nesthetized R ats with V agi I ntact and V agotomized. Rationale: The most common strategy used to simulate patient s with respiratory obstructive disease s is to apply an external re spiratory 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 vagot omy 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 a ctivated during respiratory load compensation and the neurotransmitters wit hin those neurons are still un known ITTO activated glycinergic neurons in the NTS in the first study (Chapter 2) suggest s that these NTS glycinergic neurons play potential role s in the neurogenes is of load compe nsation. Therefore, we will examine if consecutive external respiratory loads will activate glycinergic neurons in the NTS and if bilateral vagotomy will block th e expression of NTS glycinergic neurons. Hypotheses: ETTO will activate inhibitory glycinergic neurons in the NTS of anesthetized rats with vagi intact

PAGE 27

27 Vagotomy will eliminate ETTO elicited load compensation responses. Vagotomy will eliminate ETTO activated inhibitory glycinergic neurons in the NTS of anesthetized rats. Specifi c Aim 3. Identification of the L oad C ompensation R esponses and the I nhibitory N eurotransmitters in the ITTO A ctivated N eurons in the NTS of C onscious R ats. Rationale: Load compensation in conscious animals is more complex compare d 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 com pensation responses as evidence by prolonged Ti, T e 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 response s in conscious rats. Hypotheses: The pattern of ITTO elicited load compensation in con scious 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.

PAGE 28

28 Specifi c Aim 4. Identification of the E ffect of D ifferent E motional S tates on the P erception of G raded R espiratory R esistive L oads in C onscious H umans. Rationale: In conscious individuals behavioral load compensation responses are generated by the coordination of respiratory sensation, brainstem reflexes and motor functi on ( 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 Peute r 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 un clear 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 differ ent emotional states in healthy participants. The effect of different emotional state s on the perception of grated external respiratory resistive loads will be in vestigated Hypothesis : Pleasant and unpleasant emotional states will affect the perceived magnitude of respiratory resistive loads in conscious healthy participants

PAGE 29

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 )

PAGE 30

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 ANEST HETIZED RATS Background Reciprocal inhibitory connections between brainstem respiratory related neurons have been bel ieved to play important roles in shaping the r hythm 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 neu rons 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 patt ern 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 respirat ory neural network also coordinate s breathing with other respiratory related reflexes such as cough, swallow, sneeze and vomit by means of reconfiguration of the respiratory neuron al pattern in the brainstem ( Ambalavanar et al., 2004 ) Stud ies have shown that electrically stimulated cough and swallow activate neurons in the SolI

PAGE 31

31 and SolVL subnuclei of the NTS, lateral tegmental field of the reticular formation, the area postrema and the nucleus ambigu us in anesthetized cats ( Ambalavanar et al., 2004 ) Respiratory resisti ve loads have activated the neurons in the NTS, nucleus ambig u us and periaqueductal gray in anesthetized rats ( Pate and Dav enport, 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 f or the integration of respiratory, cardiovascular, gastrointestinal and gustatory peripheral afferents traveling in the facial, glossop h aryngeal 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 sens itive neurons ( Ezure and Tanaka, 2000 ) These neurons receive and process information f rom the afferents innervating the lung s and airways and make connection s with other interneurons in the NTS or project to other respi ratory 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 breath ing patter ns 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

PAGE 32

32 expiration is determined by the inspired and expired air volume and transmura l pressure across the airways, respectively ( Zechman et al., 1976 ) PSRs in the lung s and airways sense the changes in the lung volume during respiratory loading and their afferents termi nate on the p cells or other interneurons in t he NTS. Some p cells synapse with other interneuro ns 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 anesthetiz ed anim als that include d prolongation o f Ti, Te and Tt ot 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 gl ycinergic neurons. We used immuno fluorescen c e double staining of c Fos, a biomarker of activated neurons and GlyT2, a biomarker of glycinergic neurons to test our hypothesis. M aterials and Methods Animals E xperiments 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 h r light / 12 h r 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 P rocess 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

PAGE 33

33 terminated. Body temperature was monitored with a rectal temperature probe (Harvard Apparatus; Holliston, MA) and maintained at 37 39C using a heating pad. Animals were spontaneously breathing room air throughout the experiment. Animals were placed in a supine position a nd 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 a nd connected to a pressure transducer to measure esophageal pressure (Pes). The analog output s 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 l ed into an amp lifier (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 separate d 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 appl ied with the syringe inflated

PAGE 34

34 the cuff bladder to compres s the trachea causing complete closing of the airway. Removal of the pressure restored the trachea back to its original condition with no in ter ference to breathing. Experimental P rotocol 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 occlusio n 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. Animal s w ere euthanized and perfused with saline and 4% paraformaldehyde B rain s were harvested and fixed in 4% paraformaldehyde for 24 h and then transferred into a solution of 30% sucrose in PBS. The fixed brain s were frozen and sectioned coronally sections with a cryostat (HM101, Carl Zeiss; Thornwood, NY) for immunofluorescence double staining. Immunofluorescence D ouble S taining P rotocol For each brain, every fourth section of tissue was used for immun ofluorescence 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 1h r 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; Car pinteria, CA) for 36 hrs. The following day the tissue was washed three times with PBS T, and

PAGE 35

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 tissue s 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). N egative cont rol s were performed in the absen ce of the primary antibody and the results showed no c Fos or GlyT2 positive staining Data A nalysis Breathing pattern Spike 2 software (Cambridge Electronics Design; Cambridge, UK) was used for the analysis of breathing pattern. The EMGdia sign als were rectified and integrated with a time constant of 50 ms (Figure 2 1) The T i, 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 su m of Ti and Te. The EMG dia amplitude was measure d fr om 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 measure d as the difference from the baseline to the peak of P es. Ti, Te, Ttot, EMGdia amplitude and Pes of occluded and recovery breath were averaged and the n normalized by using mean values of the control breath s for each rat in experimental group.

PAGE 36

36 Immunofluorescence double s taining c Fos is a t ranscription factor located in the nucleus of a neuron. GlyT2 is a membrane protein which is a specific biomarker for glyciner gic 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 ce ll. A positive immunofluorescence double staining of c Fos and GlyT2 was charact erized 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 ) T wenty 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 o r f our levels of each region (rNTS or cNTS) in each brain were used for the immunofluorescence double stai ni ng of c Fos and GlyT2 Immunoreactive cells at three or four levels of each region of e ach animal were counted manually in bilateral rNTS and cNTS by an individual blinded to the groups. There were no stati sti cally significant differences in the numbers of positive c Fos neuron and co labeled c Fos and GlyT2 neuron s on either side of these regions so the cell counts were average d 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 us e d for immunofluorescence double staining of c Fos and GlyT2 ) to generate an estimate of the total cell counts in the entir e r NTS 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. Th e value of co labeled c Fos and GlyT2 divided by the positive c Fos cell

PAGE 37

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. O ne 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 ex perimental and sham groups. A Greenhouse Geisser correction was applied in case of violated sphericity assumptions with reported significance levels referring to corrected degree s of freedom. The significance criterion for all analyses was set at p < 0 .05. Results Breathing P attern 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 b etween C and O phases, and between O and R phases, but not between C and R phases (df = 8, F = 4.022, = 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, t racheal 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 T e contributed to a significant increase in Ttot du ring tracheal occlu sions 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

PAGE 38

38 immediately after termination of occlusions (df = 8, F= 26.883, p < 0 .001, = 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 D ouble S taining In the cNTS, there were no significant differences in the number o f c Fos and co labeled c Fos and GlyT2 cells between experimental and sham groups. There were significant differences in the percentage of c F os positive cells co labeled with GlyT2 between experimental and sham groups (df = 1, F = 2 5.502 p < 0 .01 ) The pe rcentage 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 0 0 1 ) (Figure 2 4, Figure 2 5) In the rNTS, ther e was an increasing trend in the number of c Fos cells in th e experimental group compared with the sham group (df = 1, F = 3.862, p = 0.0 97 ). There were signi fi cant difference s 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 .0 1 and df =1, F = 35.195 p < 0 .001, respectively). The number of co labeled c Fos and GlyT2 cells in the experimental group w as significantly greater than the sham group (t( 8 ) = 3, 614 p < 0. 0 1 ) The percentage of c Fos positive cells co labeled with GlyT2 was higher i n the experimental group than the sham group ( t(8) = 5.933 p < 0 .0 01 ) (Figure 2 6, Figure 2 7) In the combined rNTS and c NT S, the number of c Fos cells and co labeled c Fos and GlyT2 cells showed no significant difference s between experimental and sham groups.

PAGE 39

39 However, there were significant difference s in the percentage of c Fos positive cells co labeled with GlyT2 between experimental and sham group s (df =1, F = 34.285 p < 0 .001). The percentage of c Fos positive cells co labeled with GlyT2 was higher in the experimenta l group than the sham group (t(9 ) = 6.970 p < 0 .001) (Figure 2 8). Discussion Breathing P attern 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 contribut ed to a longer Ttot during ITTO resulting in a decreas ed breathing frequency in accordan ce with a previous study ( Pate and Dav enport, 2012b ) Pes was more negative during occlusion compare d with C and R breaths indicat ing that the tracheal cuff was inflated sufficiently to c ollapse the lumen of the trachea resulting in an increase in airway resistance. EMGdia amp litude 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 the se experiment s was the use of a tracheal occluder to intrinsically ev oke respiratory lo ad compensation. In previous stu dies 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 res istance 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 intrin sic resistance; however, also causes airway inflammation. In o ur laboratory, a tracheal occlude r was implanted a round the trachea and inflating the cuff

PAGE 40

40 increases airway resistanc e intrinsically by closing the lumen of t rachea. This strategy increases airway resistance and successfully elicits the load compensation response s without causing airway inflammation. Immunofluorescence D ouble S taining 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 signif icantly increased by ITTO demonstrat ing 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 count s ; however, the percentage of c Fos positive cells co labeled with GlyT2 were higher in the experi mental 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 physiolo gical studies have reported th at the vagus ne rves 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 se nsory information from the lung s and airways to the second order neuron s in the lateral and media l division s of NTS ( Kalia and Mesulam, 1980a ; Kalia and Sullivan, 1982 ; Kubin et al., 2006 ; Yu, 2005 ) In the present s tudy, the distribution of ITTO activated glycinergic n eurons 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

PAGE 41

41 higher order inter neurons involved in the genesis of load compe nsation response s In fact, a variety of respiratory receptors traveling through the vagus nerves te rminate in different sub nuclei of the NTS including RARs, PSRs and c fibers but only PSRs have been demonstrated to participate in the load compensation response s ( 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, in the NTS during either normal or loaded breathing for the generation of appropriate ventilation by c hanging the breathing pattern. In addition, PSRs activated second order inter neurons synapse with higher order inter neurons in the NTS and/or brainstem respirat ory neural network interneurons may involve the neurogenesis of load compensation responses as well. In this stu dy, we used c Fos as a maker for neuronal activation Although c Fos imm unostaining is a powerful technique for the study of functional pathways of differen t systems in the neural network ; onl y when neurons are activated do they express c Fos, while inhibi ted neurons do not. However c Fos immun ostaining is not able to differe ntiat e between motor and sensory pathways since mot or, 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 s tudy, we can exclude the possib ility 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 compl ex brainstem neural network configured to produce load

PAGE 42

42 compensation response s during respiratory mechanical challenge s. This study enhance s our understa nd ing of the potential role of inhibitory g lycinergic interneurons in the NTS and we propose that they may be responsible for the neurogenesis of the respiratory load compensation Vt T response.

PAGE 43

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 represent s 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

PAGE 44

44 Figure 2 1. Effect of ITTO (shadowed area) on the respiratory load compensation Physiological results during control (C3 C1) occluded (O1 O3) an d recovery (R1 R3) breath s Determination of Ti and Te are shown in the trace of integrated EMGdia

PAGE 45

45 A B C Figure 2 2. ITTO elicited the load compensation responses in anes thetized animals Normalized breath timing values during control (C3, C2, C1), occluded (O1, O2, O3) and rec overy (R1, R2, R3) breaths ( A) The relationship between Ti and breath number. ( B) The relationship between Te and breath number. ( C) The relationship between Tt ot and breath number. The indicates a significant difference, p < 0 .05 ; the ** indicat es a significant difference, p < 0 .01

PAGE 46

46 A B Figure 2 3. ITTO e ffect on EMGdia activity and Pes in anesthetized animals. Normalized EMGdia (A) and Pes (B) during control (C3, C2, C1), occluded (O1, O2, O3) and reco very (R1, R2, R3) breaths The indicates a significant difference, p < 0 .05

PAGE 47

47 Figure 2 4. ITTO activated inhibitory gl ycinergic neurons in the c NTS in anesthetized animals Immunofluorescence double staining of c Fos (red) and GlyT2 (green ) in the c NTS (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. Arrow s represent immunoreactive cells. Photo courtesy of Hsiu Wen Tsai

PAGE 48

48 A B C Figure 2 5. Immunofluorescence double staining of c Fos and GlyT2 in the c NTS in anesthetized animals (A) c Fos labele d cell number (B) co labeled c Fos and GlyT2 cell number (C) the percentage of c Fos positive cells co labeled with GlyT2 T he ** indicates a significant difference, p < 0 .01

PAGE 49

49 Figure 2 6. ITTO activated inhibitory glyc inergic neurons in the r NTS in anesthetized animals Immunofluorescence double staining of c Fos (red) and GlyT2 (green) in the r NTS (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 dash ed area in B D and H J respectively. TS: Solitary Tract. Arrows represent immunoreactive cells. Photo courtesy of Hsiu Wen Tsai

PAGE 50

50 A B C Figure 2 7. Immunofluorescence double staining of c Fos and GlyT2 in the r NTS 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 ce lls co labeled with GlyT2 The indicates a significant difference, p < 0 .05; the *** indicates a significant difference, p < 0 .001

PAGE 51

51 A B C Figure 2 8 Immunofluorescence double staining of c Fos and GlyT2 in the combined rNTS and c NTS 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 T he *** indicates a significant difference, p < .001

PAGE 52

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 i n animals to simulate the limitation of airflow in patients with respiratory obstructive disease. Respiratory load compensation is characterized by the Vt T reflex in animal s ( Clark and von Euler, 1972 ; Zechman et al., 1976 ) Inspiration and expiration were prolonged by inspiratory or expiratory loads, respect ively ( Zechman et al., 1976 ) A ugmented respiratory motor output is also characterized as load compensation response s ( Koehler and Bishop, 1979 ; Lopata et al., 1983 ) Vagal afferents have been demonstrated to trans mit 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 different ly ; b ilateral vagot omy 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 re spiratory loading of entire breath s Moreover, t he sensory and motor pathways of respiratory load compensation elicited by external re spiratory load s have been studie d extensively. T he brainstem neuron s activated during the respiratory load compensation response and the neurotransmitters within those neurons are still unknown. In the first study (Chapter 2) i t

PAGE 53

53 has been demonstrated that ITTO activated glycinergic neurons in the NTS. The results suggest that inhibitory glycinergic neurons in the NTS may p l ay a role in the neurogenesis of load compensation in anesthetized animals. In th e present study, we determine d if the load compensa tion response during sustained external respiratory loads would be abolished by bilateral vag otomy. In addition, we investigated if the gly cinergic neurons in the NTS w ould 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. M aterials an d Methods Animals E xperiments 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 h r light/12 h r da rk 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 P rocedure The rats were randomly divided into four groups (A) va gi intact rats without external and tran sient tracheal occlusions (ETTO ) (B) vagi intact rats with ETTO (C) vagotomized rats without E TTO and (D) vagotomized rats with E TTO Animals were anesthetized with urethane (1.3 g/kg, ip). Adequate anesthetic de pth 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

PAGE 54

54 maintained at 37 39C 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 (po lyethylene 50 tubing) connected to a pressure transducer to m onitor blood pressure. A saline filled tube (polyethylene 90 tubing) was passed through the mouth into the esophagus and connected to a pressure transducer to mea sure Pes The analog output s 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 change s 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 th e 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 ou tput 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 trachea l cannula was ins erted into the tracheotomy In addition, in group C and D bilateral cervical vagus nerves were separated from surrounding tissues and carotid artery and severed.

PAGE 55

55 Experimental P rotocol 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 cervic al vagotomized. The group D received 10 min of ETTO but the tracheal occlusion was not performed in group C. The occlusion was app lied for 2 3 seconds by blocking the outlet of the tracheal c annula 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 9 0 min post surgical period but did not receive obstructed breathing. A nimal s were maintaine d under anesthesia for 90 minutes following completion of the experimental protocol. Following t his 90 min period the animal s were euthanized and perfused. B rain s 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 sections with a cryostat (HM101, Carl Zeiss) for immunofluorescence double staining analysis. Immunofluorescence D ouble S taining P rotocol For each brain, every four th section of tissue was used for immun ofluorescence 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 (1:1 000 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

PAGE 56

56 following day the tissue was washed thre e 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). N egative cont rol s were performed in the a bsence of the primary antibody and the results showed no c Fos or GlyT2 posit ive staining Data A nalysis The methods are the same as described in Chapter 2. Statistical A nalysis 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 wer e 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 sig nificance levels referring to corrected degrees of freedom. The significance criterion for all analyses was set at p < 0.05. Results Breathing P attern In group A and group C there were no significant differences in Ti, Te, Ttot, EMGdia and Pes between breaths during the experiment.

PAGE 57

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, = 0.189 for Te; df = 8, F = 16.702, p < 0 .001 for T tot). 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, F igure 3 1 top panel, Figure 3 2 B ). 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 EMGdi a; df = 8, F = 3.721 p < 0.001 for Pes). Pes was more negative during occlusion s 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 3 A ). 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 = 27.13 3, p < .001 for Ttot). Tracheal occlusion s 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 5 A ). There were no s ignificant differences in EMG ia betw een C, O and R phases (Figure 3 5 B ).

PAGE 58

58 Immunofluorescence D ouble S taining Figure 3 6 and Figure 3 7 show the double stai ning of c Fos and GlyT2 in the c NTS in the four groups In the c NTS, no significant differences in the number of c Fos cells w ere observed among groups (Figure 3 8 A ). The number of co labeled c Fos and GlyT2 cells showed significant differen ces among groups (df = 3, F = 8.86 p < 0 .01 ). The number of co labele d c Fos and GlyT2 cells in group B was significant greater than group A C and D (g roup A vs. group B: t(6) = 3.961 p < 0 .01; group B vs group C: t(6) = 5. 173 p < 0 .01 ; g roup B vs group D: t (6) = 2.689 p < 0 .0 5 ) (Figure 3 8 B ). There were significant difference s 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 (g roup A vs. group B: t(6) = 6 .641 p < 0 .001; g roup B vs. group C: t(6) = 5. 061 p < 0 .01; gr oup B vs. group D: t (6) = 3. 934 p < 0 .01) (Figure 3 8 C ) but there were no significant differences between groups A, C and D F igure 3 9 and Figure 3 10 show the double stai ning of c Fos and GlyT2 in the rNTS. In the r NTS, there were no significant differences in the number of c Fos cells among the four groups (Figure 3 11 A) 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 tha n group D ( group A vs. group B: t(6) = 2.613 p < 0 .0 5 ; group B vs group C: t(6) = 2.795 p < 0 .0 5; group B vs group D : t(6) = 1.867 p = 0.111 ) The pe rcentage of c Fos and GlyT2 co labeled cells showed significant differences among the four groups (df = 3, F = 21.226 p < 0 0 01) (Figure 3 11C) The percentage of c Fos positive cells co labeled with GlyT2 was significantly greater in

PAGE 59

59 group s B than group A C and D ( group A vs. group B: t(6) = 7.270 p < 0 .0 01 ; group B vs group C: t(6) = 10.722 p < 0 .0 01 ; group B vs. group D : t(6) = 4.948 p = 0 < 0.01 ) For the combined r NTS and cNTS the number of c Fos cells showed n o significant difference among the four groups (Figure 3 12A). There were significant differences in the number of co labeled c Fos and G lyT2 cells and t he percentage of c Fos positive cells co labeled with GlyT2 (df = 3, F = 10.668 p < 0 .001 and df = 3, F = 2 3.586 p < 0 .001, respectively ) (Figure 3 12B and Figure 3 12C). The number of co labeled c Fos and G lyT2 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 (g roup A vs. group B: t(6) = 4. 396 p < 0 .01; g roup 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 (g roup A vs. group B: t(6) = 7.505 p < 0 .001; group B vs. group C: t(6) = 8.0 67, p < 0 .001; group B vs. group D: t (6) = 4. 741 p < 0 .01). Discussion Breathing P attern In group B, prolongation of Ti, Te Ttot, and increased amplitude of EMGdia du ring O phase indicates the respiratory load compensa tion 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 slight ly shorter than during C and R phase i n group D indicating that respiratory load compensation responses were suppressed by bilateral cervical vagotomy. During C and R phases, the breathing p attern in vagtomized animals was slower than animals with intact vag i. This is a typical breathing pattern in

PAGE 60

60 vagotomized animals due to the interruption of transmission of afferent activity from PSRs during inspiration to terminate inspiratory effort; therefore the ce ntral brainstem is mediating the control of breathing r hythm and pattern. In addition, the s h orter Te and Ttot during O1 compared to C and R breath s suggests o ther extravagal mechanism s such as afferents from the respiratory muscles or o the r no n vagal respiratory afferents contribute to the modulation of brea thing patterns ( Campbell et al., 1964 ; Corda et al., 1965 ; Lynne Davies et al., 1 971 ) Immunofluorescence D ouble S taining To our knowledge, this is the first s tudy demonstrating that inhibitory glycinergic ne urons in the c NTS 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 subdi visions of the NT S. These activated glycinergic neurons in the NTS might be considered as the second order interneurons possibly p c ells or p cell related higher order interneurons. Fir st, the afferents of PSRs in the lung s 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, S olI, 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 acid s on the SolM resulted in reflex termination of inspiration and prolongation of expiratio n while blockade of excitatory amino acid s in this area reduced these changes ( Bonham et al., 1993 ; Bonham and McCrimmon, 1990 ) Finally these glycinergic neurons are consistent wit h the role of

PAGE 61

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 BtC for the termination of inspiration and prolongation of expira tion and pr esynaptically inhibit inspirator y neuron s 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 c NTS and rNTS was abolis hed by bilateral cervical vagotom y Kalia et al have demonstrated that vagal afferents enter the lateral medulla from B regma 12.8 to 15.3 mm and terminate in differe nt subdivisions of the NTS from B regma 11.8 to 18.3 mm ( Kalia and Mesulam, 1980a ) In the pre sent study, the r NTS was defined as the region from B regma 12.5 to 13.3 mm and cNTS was from B regma 13.3 to 14.1 mm thus includ ing the area of entry and termination of vagal afferents. Therefore, the present results demonstrate that bilateral cervical vag otomy could successfully block transmission of information from the PSRs in the lung s and airways to glycine rgic 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. T he differe nt degree of glycinergic neuron suppression between caudal and rostral part s of the NTS m ay represent different interconnections between neurons in the brainstem control network. Therefore, furt her research needs to be pe rformed to differentiate the ro le s of the r NTS l and c NTS in controlling breathing pattern during respiratory load compensation.

PAGE 62

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

PAGE 63

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

PAGE 64

64 Figure 3 1. Effect of v agotomy on the respiratory load compensati on response s Physiological results during control (C3 C1), occluded (O1 O3) and recovery (R1 R3) breaths. Top panel : b reathing pattern in a rat with intact vagi. B ottom panel: b reathing patt ern 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

PAGE 65

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) a nd rec overy (R1, R2, R3). ( A) The relationship between Ti and breath number. ( B) The relationship between Te and breath number. ( C) The relationship between Tt ot and breath number. The indicates a significant difference, p < 0 .05

PAGE 66

66 A B Figure 3 3. ETTO e ffect on EMGdia activity and Pes in anesthetized animals with intact vagi. Normalize d 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

PAGE 67

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 rec overy (R1, R2, R3). ( A) The relationship between Ti and breath number. ( B) The relationship between Te and breath number. ( C) The relationship between Tt ot and breath number. The indicates a significant difference, p < 0 .05

PAGE 68

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

PAGE 69

69 Figure 3 6. E TTO 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 repre sent the dashed area in B D and H J respectively. TS: Solitary Tract. Arrows represent immunoreactive cells. Photo courtesy of Hsiu Wen Tsai

PAGE 70

70 Figure 3 7. The effect of v agotomy 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 c NTS (Bregma 13.8 mm ) in vagotomized animal s 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

PAGE 71

71 A B C Figure 3 8. Immunofluorescence double staining of c Fos and GlyT2 in the c NTS in anesthetized animals with or without intact vagi (A) c Fos labeled cell number (B) c o 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

PAGE 72

72 Figure 3 9. E TTO activated inhibit ory glycinergic neurons in the r NTS 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

PAGE 73

73 Figure 3 10. Effect of v agotomy on the activation of inhibitory glycinergic neurons in the rNTS in anesthetized animals Immunofluorescence staining of c Fos (red) and GlyT2 (green) in the r NTS (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

PAGE 74

74 A B C Figure 3 11. Immunofluorescence double staining of c Fos and GlyT2 in the r NTS in anes thetized animals with intact vagi and vagotomy (A) c Fos label ed 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

PAGE 75

75 A B C Figure 3 12 Immunofluorescence double staining of c Fos and GlyT2 in the combined rNTS and c NTS in anest hetized 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

PAGE 76

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 extensive ly 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 r espirato ry load compensation responses are characterized as Vt T relationship s in anesthetized animals. Applying a single inspiratory or expiratory load decreased Vi and Ve and resulted in a prolongation of Ti and Te respectively ; t he timing paramete rs were not affected during unloaded breaths ( Clark and von Euler, 1972 ; Zechman et al., 1976 ) This is a vagal dependent re flex and mediated by PSRs in lung s and airways. PSRs respon d to changes in lung volume or transmural pressure across t he 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 increa se of respiratory motor outputs, including diaphragm and abdominal muscle activity ( Koehler and Bishop, 1979 ; Lopata et al., 1983 ) In anesthetized animals, the load compensation response s are mediated by the respiratory neurons in the brainstem. In conscious animals, r espiratory load compensation involve s cortical (primary sensorimotor cortices, supplementary motor and premotor cortex ) and subcortical neural structure s (thalamus, globus pallidus caudate, cerebellum and limbic system ) to

PAGE 77

77 voluntar il y modify breathing p attern ( 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 t halamus determines if a respiratory load will be gated in or out of the cortical and sub corical 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 ( d iscriminative 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 d iscri m inative dimension is rela ted 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 m otor cortex receive s the integrated sensory information and send s projec t ions 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 expi ratory load by dilating the larynx and prolonging the Te followed by a return to bas eline 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 augment ation of diaphragm activity during the

PAGE 78

78 first loaded b reath and only had minimal changes after first loaded breath ( Fo rster 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 strength s load duration s load applications, specie s and age. P revious studies used external respiratory load s 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, r espiratory load compensat ion elicited by sustained intrinsic tracheal occlusion s in consciousness has not been studied. In our laboratory, we developed a new surgical strategy to produce intrinsic, tra nsient tracheal occlusion without changing lung compliance. The first purpose of the present study is to determine the ITTO elicited load compensation response s in conscious animals. Additionally, respiratory drive in anesthetized animal is mainly dominated by the reciprocal interconnections with in the brainstem neural network. In t he model proposed by Rybak ( Rybak et al., 2007 ; Rybak et al., 2008 ) the brainstem respiratory neural network c onsist s of synaptic connection s between the pons, ventral medulla and NTS Inhibitory reciprocal intercon nection s between the pos t I, aug E in the BtC and pre I, early I in the pre BtC are proposed to generate the respiratory rhythm. The pons has excitatory projection s to the respiratory neurons in the dorsal and ventral medulla to modulate the rhythm and pattern of breathing. The NTS is the site of i ntegrating and processing respiratory peripheral afferents from lung s 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 lung s and airways are the major

PAGE 79

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 critical ly important for the neurogenesis o f 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 b reathing pattern i s modulated by cortical and subcotical st r uctures implying that consciousness and behavior are important for the neurogenesis of conscious load compensation responses. Ther efore, the second purpose of th is study is to determine if the glycinergic neurons i n the NTS are activated in the neurogenesis of the load compensation response s in conscious animal s We hypothesized that glycinergic neur ons in the NTS would be less activated by ITTO in conscious animals compared to anesthetized animals. We used immunofl uorescence double staining o f c Fos and GlyT2 to test our hypothesis. Materials and Methods Animals E xperiment s w ere performed on 11 male Sprague Dawley rats ( 320 380g ) The animals were housed in the University of Florida animal care facility. They w ere exposed to a 12 h r light/12 h r 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 P rocedure Animals were anesthetized using inhaled isoflurane gas (2 5% in O 2 ). Adequate anesthetic depth was verified by the absence of a withdrawal reflex to a rear paw pinch.

PAGE 80

80 Buprenorphine (0.04 0.05 mg/kg body weight) and carprofen (5mg/kg body weight) w ere administered preop eratively via subcutaneous injection. The eyes were coated with petroleum ointment to prevent drying. Incision sites w ere shaved and sterilized with povidine iodine topical antiseptic solution. Body temperature was monitored with a rectal probe and maintai ned at 37 39C 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 v entral incision was closed using an interrupter suture pattern. Following surgery, rats w ere 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 P rotocol The Sprague Dawley rats (320 380 g) w ere 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, follow ed by 10 minutes of ITTO then 90 min post occlusion eupne ic 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 animal s w ere euthan ized and perfused. B rain s were harvested and fixed in 4%

PAGE 81

81 paraformaldehyde for 24 h and then transfer into a solution of 30% sucrose in PBS. The Zeiss) for immunofluorescence analysis. Immunofluorescence D ouble S taining P rotocol The double staining methods are the same as described in Chapter 3 Data Analysis The data analysis methods are the same as des cribed in Chapter 2 Statistical A nalysis The statistical ana lyses are the same as described in Chapter 2 Results Breathing P attern Figure 4 1 illustrates the breathing pattern in conscious animals. T here 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 wh en compared to C and R phases ( df = 8, F = 10.876, p < 0 .00 1 ) ( 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 D ouble S taining In the cNTS, there were no significant differences in the n umber of c Fos cells, co labele d 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).

PAGE 82

82 In the rNTS (Figure 4 6) there w ere no significant d ifference in the number of c Fos cells and co labeled c Fos and Gly T2 cells between these two groups (Figure 4 7A, Fig ure 4 7B). The percentage of c Fos positive cells co labeled with GlyT2 showed significant differences betwe en two groups (df = 1, F= 1 2.647 p < 0 .01) (Figu re 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 combine d r NTS l and c NTS, 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 w as a significant dif ference in the percentage of c Fos positive cells co labeled with GlyT2 betwe en two groups (df = 1, F = 9.544 p < 0 .05). The percentage of c Fos positive cells co labeled with GlyT2 was significantly greater i n the experimental group than the sham group ( t(9) = 3.089 p < 0 .05 ) (Figure 4 8C). Discussio n Breathing P attern The load compensation re sponses in conscious animals were found t o 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, c onscious animals behavi orally prolonged Te in response to ITTO without changing Ti The prolongation of Te contri butes to the increase of Ttot. During occlusion s conscious rats did not respond to ITTO in the beginning of the occlusions (O1 and O2), but behaviorally increase d their expirati on only by O3 and then return ed to normal breathing immediately after the removal of occlusions demonstrating that the load compensation response in conscious animals might be affected by consciousness

PAGE 83

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 load s during one phase of breathing (inspiration or expiration) or one entire breath. However, i n the present study, consecutive sustained respiratory loads to both inspiration and expiration were presented in conscious animals for up to 2 3 sec th at could cause an aversive sensation If a n aversive sensation was elicited with the ITTO then the cons cious 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 lo ad compensation responses in this study whereas previous studies used external respiratory load s 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 intrinsica lly without changing lung compliance. Immunofluorescence D ouble S taining ITTO elicited the activation of g ly cinergic neurons in the r NTS but not in the c NTS 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. The se results support our hypothesis that the NTS play s 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 t he SolM and SolV is consistent with the previous stud ies that ITTO and ETTO activated glycinergic neurons in the same subdivisions of NT S in anesthetized animals The activation of glyciergic neurons in the SolVL was only seen in conscious animals but not

PAGE 84

84 in anesthetized animals. It has been reported that application of excitatory amino aci d s on the SolM containing p cells caused reflex termination of inspiration and prolongation of expiration while blockade of excitatory amin o acid in this area reduced th ese 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 n eurons in the SolM mainly project to the ventrolateral medulla, parafacial region and rVR G and only few SolM neurons sen d projections to the BtC and pre BtC ( Alheid et al., 2011 ) The projections from SolVL to the VRC are more broad ly distributed compared to other subdivisions of NTS ( Alheid et al., 2011 ) Therefore, the activated glycinergic neurons in the SolM in both consciou s and anesthetized animals may be the primary neural area responsible for the generation of load compensation respon ses. Additionally the different distribution of proj ections from the SolM and SolVL might mediate difference mechanisms to modulate breathing pattern. In the present study, activated glycinergic neu rons 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 lung s and airways and send projection to the VRC and pons for the modulation of breathing pattern. It has been demon strated that two thirds of p cells in the NTS use GABA and glycin e as neurotransmitters ( Ezure and Tanaka, 2004 ) Although we saw the elevated expression of glycinergic neurons in the SolM and SolV in conscious an imals as we found in anesthetized animals, the patterns of load compensation responses are different between consci ous and anesthetized states

PAGE 85

85 suggesting that other mechanisms play more impor tant roles in the behavioral load compensation. SolVL is a candid ate brain region for the neurogenesis of load compensation in c onscious animals since glycinergic neurons in the SolVL are only activated in conscious animals. As noted previou sly the SolVL is n ot necessary for reflex load compensation. S everal studies have demonstrated that the SolVL receive s projections fro m cortical and subcortical structures such as subcor tical cortex ( van der Kooy et al., 1984 ) and the central nucleus of amygdal a ( Danielsen et al., 1989 ) The activated glycin ergic 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 l oad compensati on responses with sustained respiratory loads in conscious animals. Based on the se results, cortical and subcortical processing of respiratory load sensation override s brainstem reflexes for conscious modification of breathing pattern.

PAGE 86

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

PAGE 87

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) b reaths Determination of Ti and Te are shown in the trace of integrated EMGdia. Shadow area represents the period of tracheal occlusion s

PAGE 88

88 A B C Figure 4 2. Effect of s ustained 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 Tt ot and breat h number. The indicates a significant difference, p < 0 .05

PAGE 89

89 Figure 4 3. Sustained ITTO e ffect 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

PAGE 90

90 Figure 4 4. The effect of ITTO on the activation of i nhibitory glycinergic neurons in the cNTS in conscious animals. Immunofluorescence double staining of c Fos (red) and GlyT2 (green) in the c NTS (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 re present immunoactiv e cells. Photo courtesy of Hsiu Wen Tsai

PAGE 91

91 A B C Figure 4 5. Immunofluorescence double staining of c Fos and GlyT2 in the c NTS in conscious animals (A) c Fos labele d cell number (B) co labeled c Fos and GlyT2 cell number (C) the percentage of c Fos positive cells co labeled with GlyT2

PAGE 92

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 i n B D and H J, respectively. TS: Solitary Tract. Arrows represent immunoactive cells. Photo courtesy of Hsiu Wen Tsai

PAGE 93

93 A B C Figure 4 7. Immunofluorescence double staining of c Fos and GlyT2 in the r NTS in conscious animals (A) c Fos labele d 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 .0 1

PAGE 94

94 A B C Figure 4 8. Immunofluorescence double staining of c Fos and GlyT2 in the combined r NTS and c NTS in conscious animals (A) c Fos labele d cell number (B) co labeled c Fos and Gl yT2 cell number (C) the percentage of c Fos positive cells co labeled with GlyT2 The indicates a significant difference, p < 0 .05

PAGE 95

95 CHAPTER 5 THE IMPACT OF EMOTION ON THE PERCEPTION OF GRADED MAGNITUDES OF RESPIRATORY RESISTIVE LOADS Background Breathing i s 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 processi ng 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 emotion al 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 ove ruse of

PAGE 96

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 a ffects the perception of respiratory sensations regardless of physiological ventilatory changes. These studies have shown that patients with COPD or asth ma 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 aff ective 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 pleas ant 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 com pensation 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.

PAGE 97

9 7 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 established method of affective picture view ing ( Lang, 2008 ) Materials and Methods Participants The study was approved by the Institutional Review Board of the University of Florida. Twent y four healthy adults with no history of pulmonary or neurological disease participated i n the study after providing informed written consent. To ensure vital baseline lung function, participants underwent standard spirometry prior to testing. Baseline cha racteristics of the participants are listed in Table 5 1. Affective P icture S eries 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 R esistive L oads The resistive loading manifold was placed at the d istal part of a breathing circuit and consisted of 5 magnitudes of resistive loads (5, 10, 15, 20, 45 cmH 2 O/L/sec) and no load. Resistive loads were sintered bronze disks placed in series in the loading manifold and separated by stoppered ports. The resist ances were presented for a single inspiration by removing a stopper from one selected port and allowing the subject to

PAGE 98

98 inspire through the load. Following each load presentation, participants estimated the perceived difficulty of breathing using the modifi ed Borg scale which ranges from 0 (= nothing at all) to 10 (=very, very serve) ( Borg, 1982 ) Measurement of R espiration 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, C O, USA). Measurement of E motional R esponses 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 rat ings 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 presen tations 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

PAGE 99

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 A nalysis 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 load s (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 as sumptions 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 ) (pleasant vs. neutral:

PAGE 100

100 t(23) = 6.20, p < 0 .001; unpleasant vs. neutral: t(23) = 8.41 p < 0 .001; u npleasant vs. pleasant: t(23) = 9.50, p < 0 .001). Arousal ratings showed significant dif ferences between the con ditions ( 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 observe d between load and condition ( df = 8, F = 2.441, p < 0 .05 ) Post hoc test showed that for the 5 cmH 2 O/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 cmH 2 O/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

PAGE 101

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 cond ition. 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 cmH 2 O/L/sec. With resistive loads greater than 5 cmH 2 O/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 cmH 2 O/L/sec load. This decrease in slope iratory 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.

PAGE 102

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, th ese 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 rathe r sma ll 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 red uces or has no

PAGE 103

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 functio n 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 de monstrate an emotional modulation of respiratory perception at higher res istive 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 r esistive 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 combin ation 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

PAGE 104

104 Table 5 1. Baseline characteristics of participants (Mean, SD) Characteristics Data Age (y ea rs) 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)

PAGE 105

105 Figure 5 1. Schematic of e xperimental s etup

PAGE 106

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

PAGE 107

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

PAGE 108

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

PAGE 109

109 Figure 5 5. Mean of slope of LogME LogPmax for the pleasant, neutral and unpleasant picture series. Error bars represen t standard errors of the mean. p < 0.05

PAGE 110

110 CHAPTER 6 SUMMARIES AND CONCLUSIONS Summary of Study Findings Study #1 Summary Respirato ry load compensation is a senso ry motor reflex generated in the brainstem respiratory neural network. The NTS is thought to be the primary struc ture to process the respiratory load related afferent activity and contribute to the modification of breathing pattern by sending efferent projections to o ther structures in the brainstem respiratory neural network, such as the VRC and PRG. The sensory pathway and motor responses of r espiratory load compensation ha ve been extensively studied; however, the mechanism of neurogenesis of load compensation is still unknown A variety of studies have shown that inhibitory intercon nections 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 neuron s in the NTS were activated by ITTO in anesthetized animals. The results showed that load compensation responses including prolonged Ti, Te and Ttot were el icited by ITTO. In addition, glycinergic neurons in both r NTS and c NTS were activated in anesthetized animals with I TTO compared with the sham group. T he 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 glyci nergic neurons in the NTS as found in the first study (Chapter 3) In addition we investigated the effect of v agotomy on the respiratory load co mpensation response s

PAGE 111

111 and glycinergic neuron exp ression in the NTS. The results showed that ETT O produce d load compensation responses wit h incr eased Ti, Te, Ttot as well as elevated activation of inhibitory gly cinergic 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 C onscious animals respond to respiratory load s differe ntly tha n anesthetized animals due to cortic al and subcortical processing. C onscious animals behaviorally contr ol breathing pattern in response to respiratory challenge s. Therefore, we hypothesized that the mechanism of neurogenesis of load compensation in the brainstem in conscio us animals would differ from anesthetized animals. In the present study, we examined the load compensation responses in conscious animals with sustained respiratory loads. In additio n, we determine d if inhibitory glycinergic neurons would be activated in the NTS in conscio us animals as we found in anesthetized animals The results demonstrated that conscious animals responded to ITTO with pro longation of Te and Ttot not Ti which is different from anesthetized animals. Moreover, g lycinergic neuron s were activated in the r NTS but not in the c NTS. These results suggest that the activated glycinergic neurons in the r NTS are more important for the neurogenesis of load compensation responses than c NTS 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 percep tion of respiratory loads of different load magnitudes. Twenty four healthy adults

PAGE 112

112 participated in the study. Five inspiratory resistive loads of increasing magnitude (5, 10, 15, 20, 45 cmH 2 O/L/sec) were repeatedly presented for one inspiration while parti cipants 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 subje cts 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 Loa d Compensation in Anesthetized and Conscious Animals I ndividual s are able to compensate for respiratory challenges by modify ing 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 significant ly prolong ation of Ti, Te and Ttot consistant with previous studies ( Bernhar dt et al., 2011a ; Pate and Davenport, 2012b ) During the O phase, Ti was significantly i ncreased only during O 1 whereas Te was prolonged during the entire occluded period This m ay b e 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 t o initiate ITT O at specific breath phases with accurate timin g especially during inspiration as it is short er than expiration I n addition, acute ITTO did not enhance inspiratory muscle outputs in the present study whereas c hronic ITTO co nditioning has been

PAGE 113

113 shown to be associated with type II fiber hypertrophy in the medial costal region of the diaphragm ( Smith e t al., 2012 ) This indicate s that acute ITT O is a good model to study respiratory load compensa tion responses and chronic ITTO might be a good animal model for clinical respiratory muscle strength training, incl uding individuals with st r oke or spinal co rd injury or weaning patients from mechanical ventilator s In conscious animals, r epetitive application of acute ITTO might cause aversive respiratory sensation s We have observed that c onscious animals w ill hold their breathing during expiration instead of breath ing through the occlusion loads presumably to alleviate the aversive sensation which is a fear escape response. P revious stud ies ha ve demonstrated that c hronic ITTO conditioning change d gene expre ssion in the medial thalamus an integral region involved in the processing of respiratory sensory pathway ( Bernhardt et al., 2011b ) and ITTO conditioning caus ed stress, anxiety depression and neural state changes ( Pate and Davenport, 2012a ) In the present study, conscious rats showed t he fear escape response in the face of acute ITTO which may ultimately evolve into depress ion and anxiety. In addition s tudies have shown that pati ents with respiratory obstructive diseases often present with affective disorders, such as anxiety and /or panic ( Brenes, 2003 ; Wagena et al., 2005 ) H owever, the reason s for the expression of affective disorders in respira tory obstructed patients are still not clear Base d on the present and previous studies, we suggest that animals that experience repeated respiratory obstructio n will behaviorally modify their breathing pattern to avoid aversive respiratory sensation s and eventually develop affective disorders due to chronic res piratory obstructed breathing

PAGE 114

114 Effect of Emotion on R espiratory P erception Acute ITTO in conscious rats elicit ed behavioral fear escape response (Chapter 4 ) and chronic ITTO conditio n ing c ause d anxiety and panic ( Pate and Davenport, 2012a ) Studies have shown that negative affect ( NA ) affected the a ccuracy of respiratory perception in patients with respiratory obstructive dise ases regardless of their respiratory function. For example, COPD or asthmatic patien ts with higher N A reported more intense symptoms compared to patients with lower NA ( De Peuter et al., 2008 ; Vogele and von Leupoldt, 2008 ) O ver perception of r espiratory loading m ay caus e 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 respir atory load s, i t is still unknown whether emotional modulation of respiratory percep tion is different at varying l evels of respiratory restriction. The results of s tudy 4 (Chapter 5) demonstrated that picture elicited NA state increased the perceived respiratory effort for low magnitude resistive load s but not for high magnitude loads. This suggest s that respiratory perce ption in patient s with mild COPD or asthma may have less modulat ion by NA state than patients with severe CO PD or asthma since the phys iological discomfort override s the emotional impact. Activation o f Inhibitory Glycinergic Neurons i n t he NTS I n anesthetized animals, ITTO activated inhibitory glycinergic neurons in both of the caudal and ros tral NTS I n conscious animals, inhibitory glycinergic neurons were only activated in the r NTS. The differences in the results indicate that load compensation responses may be processing differently in anesthetized and conscious animals.

PAGE 115

115 In anesthetized animal s ITTO or ETTO activated glycinergic neurons were concentrated in the SolM and So lV subdivisions of the NTS. In conscious animal s glycinergic neurons were activated by ITTO in the SolM, SolV and SolVL The different subdivisions of the NTS play different rol es in the control of breathing. It has been demonstrated that interneurons in the SoM are associated with the Herin g Breuer reflex. Excitation of inter neurons 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 contras t deletion of SolVL did not impair t his respiratory reflex ( McCrimmon and Alheid, 2004 ) In addition, inter neurons in the SolM send projections to the rostral VRC, including BtC and P re BtC but interneurons in the SolVL project bro adly to the VRC ( Alheid et al., 2011 ) It has been demonstrated that interneurons in the SolVL receive projections from cortical and subcorti cal structures ( Danielsen et al., 1989 ; van der Kooy et al., 1984 ) Therefore, we proposed that these activated i nhibitory 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 c ontrol 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 li mitation is mostly the result of an increased airway resista nce (airway obstruction) act ing 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

PAGE 116

116 cha nging breathing patterns to sustain alveolar ventilation which is respiratory load compensation. Respiratory l oad compensation is a sensory motor reflex resul ting 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 loa d 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 obs tructive 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 ( C hapter 2) and 2 ( C hapter 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 ge nerating the load compensation responses ( Rybak et al., 2007 ; Rybak et al., 2008 ) S tudy 3 (Chapte r 4) and 4 ( C hapter 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 s tate s would change perceived respiratory effort and add behavioral modulation to the load compensation responses. These findings advance and expand ou r knowledge for the modeling of the respiratory neu ral network in the brainstem as well as cortical and subcortical regions.

PAGE 117

117 Direction for Future Studies Futu re studies are envisioned to combine a multi electrode array recording system s and computational methods to investigate the cross correlation between neurons in the NTS and VRC during respiratory mechanical load s We aim to demonstrate the presence of an inhibitory projection from the NTS to the VRC. Conclusions This dissertation examine d 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 respir atory effort in humans The activated interneurons in the NTS were found to be inhibitory glycinergic in both anesth etized 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

PAGE 118

118 LIST OF REFERENCES Affleck, G., Apter, A., Tennen, H., Reisine, S., Barrows, E., Willard, A., Unger, J., ZuWallack, R., 2000. Mood states associated with transitory changes in asthma symptoms and peak expiratory flow. Psychosom Med 62, 61 68. Alheid, G.F., Jiao, W., McCrimmon, D.R., 2011. Caudal nuclei of the rat nucleus of the solitary tract differentially innervate respiratory compartments within the ventrolateral medulla. Neuroscience 190, 207 227. Alheid, G.F., McCrimmon, D.R., 2008. The chemical neuroanatomy of breathing. Respir Physiol Neurobiol 164, 3 11. Altose, M., Cherniack, N., Fishman, A.P., 1985. Respiratory sensations and dyspnea. J Appl Physiol 58, 1051 1054. Ambalavanar, R., Tanaka, Y., Selbie, W.S., Ludlow, C.L., 2004. Neuronal activation in the medulla oblongata during selective elicitation of the laryng eal adductor response. J Neurophysiol 92, 2920 2932. Araki, T., Yamano, M., Murakami, T., Wanaka, A., Betz, H., Tohyama, M., 1988. Localization of glycine receptors in the rat central nervous system: an immunocytochemical analysis using monoclonal antibody Neuroscience 25, 613 624. Arita, H., Kogo, N., Koshiya, N., 1987. Morphological and physiological properties of caudal medullary expiratory neurons of the cat. Brain Res 401, 258 266. Axen, K., Haas, S.S., 1979. Range of first breath ventilatory response s to added mechanical loads in naive men. J Appl Physiol 46, 743 751. Ballantyne, D., Richter, D.W., 1984. Post synaptic inhibition of bulbar inspiratory neurones in the cat. J Physiol 348, 67 87. Ballantyne, D., Richter, D.W., 1986. The non uniform charac ter of expiratory synaptic activity in expiratory bulbospinal neurones of the cat. J Physiol 370, 433 456. Barnes, P.J., 1994. Blunted perception and death from asthma. N Engl J Med 330, 1383 1384. Berger, A.J., 1977. Dorsal respiratory group neurons in th e medulla of cat: spinal projections, responses to lung inflation and superior laryngeal nerve stimulation. Brain Res 135, 231 254. Bernhardt, V., Garcia Reyero, N., Vovk, A., Denslow, N., Davenport, P.W., 2011a. Tracheal occlusion modulates the gene expre ssion profile of the medial thalamus in anesthetized rats. J Appl Physiol 111, 117 124.

PAGE 119

119 Bernhardt, V., Hotchkiss, M.T., Garcia Reyero, N., Escalon, B.L., Denslow, N., Davenport, P.W., 2011b. Tracheal occlusion conditioning in conscious rats modulates gene expression profile of medial thalamus. Front Physiol 2, 24. Bianchi, A.L., Denavit Saubie, M., Champagnat, J., 1995. Central control of breathing in mammals: neuronal circuitry, membrane properties, and neurotransmitters. Physiol Rev 75, 1 45. Bogaerts, K. Notebaert, K., Van Diest, I., Devriese, S., De Peuter, S., Van den Bergh, O., 2005. Accuracy of respiratory symptom perception in different affective contexts. J Psychosom Res 58, 537 543. Bonham, A.C., 1995. Neurotransmitters in the CNS control of breat hing. Respir Physiol 101, 219 230. Bonham, A.C., Coles, S.K., McCrimmon, D.R., 1993. Pulmonary stretch receptor afferents activate excitatory amino acid receptors in the nucleus tractus solitarii in rats. J Physiol 464, 725 745. Bonham, A.C., McCrimmon, D. R., 1990. Neurones in a discrete region of the nucleus tractus solitarius are required for the Breuer Hering reflex in rat. J Physiol 427, 261 280. Borg, G.A., 1982. Psychophysical bases of perceived exertion. Med Sci Sports Exerc 14, 377 381. Bradley, M.M ., Lang, P.J., 1994. Measuring emotion: the Self Assessment Manikin and the Semantic Differential. J Behav Ther Exp Psychiatry 25, 49 59. Bradley M.M.L., P. J., 2007. The International Affective Picture System (IAPS) in the study of emotion and attention ., in: (Eds.), I.J.A.C.J.J.B.A. (Ed.), Handbook of emotion elicitation and assessment. Oxford University Press, New York, pp. 29 46. Brenes, G.A., 2003. Anxiety and chronic obstructive pulmonary disease: prevalence, impact, and treatment. Psychosom Med 65, 963 970. Calabrese, P., Dinh, T.P., Eberhard, A., Bachy, J.P., Benchetrit, G., 1998. Effects of resistive loading on the pattern of breathing. Respir Physiol 113, 167 179. Campbell, E.J., Dickinson, C.J., Howell, J.B., 1964. The Immediate Effects of Added Loads on the Inspiratory Musculature of the Rabbit. J Physiol 172, 321 331. Chan, P.Y., Davenport, P.W., 2008. Respiratory related evoked potential measures of respiratory sensory gating. J Appl Physiol 105, 1106 1113. Chitravanshi, V.C., Sapru, H.N., 199 5. Chemoreceptor sensitive neurons in commissural subnucleus of nucleus tractus solitarius of the rat. Am J Physiol 268, R851 858.

PAGE 120

120 Clark, F.J., von Euler, C., 1972. On the regulation of depth and rate of breathing. J Physiol 222, 267 295. Cohen, M.I., Feld man, J.L., 1984. Discharge properties of dorsal medullary inspiratory neurons: relation to pulmonary afferent and phrenic efferent discharge. J Neurophysiol 51, 753 776. Cohen, M.I., Piercey, M.F., Gootman, P.M., Wolotsky, P., 1974. Synaptic connections be tween medullary inspiratory neurons and phrenic motoneurons as revealed by cross correlation. Brain Res 81, 319 324. Corda, M., Eklund, G., Von, E., 1965. External Intercostal and Phrenic Alpha Motor Responses to Changes in Respiratory Load. Acta Physiol S cand 63, 391 400. Danielsen, E.H., Magnuson, D.J., Gray, T.S., 1989. The central amygdaloid nucleus innervation of the dorsal vagal complex in rat: a Phaseolus vulgaris leucoagglutinin lectin anterograde tracing study. Brain Res Bull 22, 705 715. Davenport P.W., Chan, P.Y., Zhang, W., Chou, Y.L., 2007. Detection threshold for inspiratory resistive loads and respiratory related evoked potentials. J Appl Physiol 102, 276 285. Davenport, P.W., Frazier, D.T., Zechman, F.W., Jr., 1981a. The effect of the resist ive loading of inspiration and expiration on pulmonary stretch receptor discharge. Respir Physiol 43, 299 314. Davenport, P.W., Freed, A.N., Rex, K.A., 1984. The effect of sulfur dioxide on the response of rabbits to expiratory loads. Respir Physiol 56, 35 9 368. Davenport, P.W., Lee, L.Y., Lee, K., Yu, L.K., Miller, R., Frazier, D.T., 1981b. Effect of bronchoconstriction on the firing behavior of pulmonary stretch receptors. Respir Physiol 46, 295 307. Davenport, P.W., Vovk, A., 2009. Cortical and subcortic al central neural pathways in respiratory sensations. Respir Physiol Neurobiol 167, 72 86. Davenport, P.W., Wozniak, J.A., 1986. Effect of expiratory loading on expiratory duration and pulmonary stretch receptor discharge. J Appl Physiol 61, 1857 1863. de Castro, D., Lipski, J., Kanjhan, R., 1994. Electrophysiological study of dorsal respiratory neurons in the medulla oblongata of the rat. Brain Res 639, 49 56. De Peuter, S., Lemaigre, V., Van Diest, I., Van den Bergh, O., 2008. Illness specific catastrophi c thinking and overperception in asthma. Health Psychol 27, 93 99. Dutschmann, M., Paton, J.F., 2002a. Glycinergic inhibition is essential for co ordinating cranial and spinal respiratory motor outputs in the neonatal rat. J Physiol 543, 643 653.

PAGE 121

121 Dutschman n, M., Paton, J.F., 2002b. Influence of nasotrigeminal afferents on medullary respiratory neurones and upper airway patency in the rat. Pflugers Arch 444, 227 235. Elekes, I., Patthy, A., Lang, T., Palkovits, M., 1986. Concentrations of GABA and glycine in discrete brain nuclei. Stress induced changes in the levels of inhibitory amino acids. Neuropharmacology 25, 703 709. Ezure, K., 1990. Synaptic connections between medullary respiratory neurons and considerations on the genesis of respiratory rhythm. Prog Neurobiol 35, 429 450. Ezure, K., Manabe, M., 1988. Decrementing expiratory neurons of the Botzinger complex. II. Direct inhibitory synaptic linkage with ventral respiratory group neurons. Exp Brain Res 72, 159 166. Ezure, K., Manabe, M., Yamada, H., 1988 Distribution of medullary respiratory neurons in the rat. Brain Res 455, 262 270. Ezure, K., Tanaka, I., 2000. Identification of deflation sensitive inspiratory neurons in the dorsal respiratory group of the rat. Brain Res 883, 22 30. Ezure, K., Tanaka, I., 2004. GABA, in some cases together with glycine, is used as the inhibitory transmitter by pump cells in the Hering Breuer reflex pathway of the rat. Neuroscience 127, 409 417. Ezure, K., Tanaka, I., Kondo, M., 2003a. Glycine is used as a transmitter by decrementing expiratory neurons of the ventrolateral medulla in the rat. J Neurosci 23, 8941 8948. Ezure, K., Tanaka, I., Saito, Y., 2003b. Activity of brainstem respiratory neurones just before the expiration inspiration transition in the rat. J Physiol 547, 629 640. Ezure, K., Tanaka, I., Saito, Y., Otake, K., 2002. Axonal projections of pulmonary slowly adapting receptor relay neurons in the rat. J Comp Neurol 446, 81 94. Feldman, J.L., Del Negro, C.A., 2006. Looking for inspiration: new perspectives on respiratory rhythm. Nat Rev Neurosci 7, 232 242. Feldman, J.M., McQuaid, E.L., Klein, R.B., Kopel, S.J., Nassau, J.H., Mitchell, D.K., Wamboldt, M.Z., Fritz, G.K., 2007. Symptom perception and functional morbidity across a 1 year follow up in pediatric as thma. Pediatr Pulmonol 42, 339 347. Finley, J.C., Katz, D.M., 1992. The central organization of carotid body afferent projections to the brainstem of the rat. Brain Res 572, 108 116.

PAGE 122

122 Fong, A.Y., Stornetta, R.L., Foley, C.M., Potts, J.T., 2005. Immunohistochemical localization of GAD67 expressing neurons and processes in the rat brainstem: subregional distribution in the nucleus tractus solitarius. J Comp Neurol 493, 274 290. Forster, H.V., Lowry, T.F., Pan, L.G., Erickson, B.K., Korducki, M.J., Forster, M.A., 1994. Diaphragm and lung afferents contribute to inspiratory load compensation in awake ponies. J Appl Physiol 76, 1330 1339. Haji, A., Remmers, J.E., Connelly, C., Takeda, R., 1990. Effects of glycine and GABA on bulbar respiratory neurons of cat. J Neurophysiol 63, 955 965. Haji, A., Takeda, R., Remmers, J.E., 1992. Evidence that glycine and GABA mediate postsynaptic inhibition of bulbar respiratory neurons in the cat. J Appl Physiol 73, 2333 2342. Haxhiu, M.A., Kc, P., Moore, C.T., Acquah, S.S., Wilson, C.G., Zaidi, S.I., Massari, V.J., Ferguson, D.G., 2005. Brain stem excitatory and inhibitory signaling pathways regulating bronchoconstrictive responses. J Appl Physiol 98, 1961 1982. Hayashi, F., Lipski, J., 1992. The role of inhibitory ami no acids in control of respiratory motor output in an arterially perfused rat. Respir Physiol 89, 47 63. Hogg, J.C., 2004. Pathophysiology of airflow limitation in chronic obstructive pulmonary disease. Lancet 364, 709 721. Hudgel, D.W., Cooperson, D.M., K insman, R.A., 1982. Recognition of added resistive loads in asthma: the importance of behavioral styles. Am Rev Respir Dis 126, 121 125. Hutt, D.A., Parisi, R.A., Edelman, N.H., Santiago, T.V., 1991. Responses of diaphragm and external oblique muscles to f low resistive loads during sleep. Am Rev Respir Dis 144, 1107 1111. Iizuka, M., 1999. Intercostal expiratory activity in an in vitro brainstem spinal cord rib preparation from the neonatal rat. J Physiol 520 Pt 1, 293 302. Janssens, T., Verleden, G., De Pe uter, S., Van Diest, I., Van den Bergh, O., 2009. Inaccurate perception of asthma symptoms: a cognitive affective framework and implications for asthma treatment. Clin Psychol Rev 29, 317 327. Jentsch, T.J., Stein, V., Weinreich, F., Zdebik, A.A., 2002. Mo lecular structure and physiological function of chloride channels. Physiol Rev 82, 503 568. Jiang, C., Lipski, J., 1990. Extensive monosynaptic inhibition of ventral respiratory group neurons by augmenting neurons in the Botzinger complex in the cat. Exp B rain Res 81, 639 648.

PAGE 123

123 Julius, S.M., Davenport, K.L., Davenport, P.W., 2002. Perception of intrinsic and extrinsic respiratory loads in children with life threatening asthma. Pediatr Pulmonol 34, 425 433. Kalia, M., Mesulam, M.M., 1980a. Brain stem projecti ons of sensory and motor components of the vagus complex in the cat: I. The cervical vagus and nodose ganglion. J Comp Neurol 193, 435 465. Kalia, M., Mesulam, M.M., 1980b. Brain stem projections of sensory and motor components of the vagus complex in the cat: II. Laryngeal, tracheobronchial, pulmonary, cardiac, and gastrointestinal branches. J Comp Neurol 193, 467 508. Kalia, M., Sullivan, J.M., 1982. Brainstem projections of sensory and motor components of the vagus nerve in the rat. J Comp Neurol 211, 24 8 265. Kelsen, S.G., Prestel, T.F., Cherniack, N.S., Chester, E.H., Deal, E.C., Jr., 1981. Comparison of the respiratory responses to external resistive loading and bronchoconstriction. J Clin Invest 67, 1761 1768. Kifle, Y., Seng, V., Davenport, P.W., 199 7. Magnitude estimation of inspiratory resistive loads in children with life threatening asthma. Am J Respir Crit Care Med 156, 1530 1535. Killian, K.J., Mahutte, C.K., Campbell, E.J., 1981. Magnitude scaling of externally added loads to breathing. Am Rev Respir Dis 123, 12 15. Koehler, R.C., Bishop, B., 1979. Expiratory duration and abdominal muscle responses to elastic and resistive loading. J Appl Physiol 46, 730 737. Koizumi, H., Smith, J.C., 2008. Persistent Na+ and K+ dominated leak currents contribut e to respiratory rhythm generation in the pre Botzinger complex in vitro. J Neurosci 28, 1773 1785. Kubin, L., Alheid, G.F., Zuperku, E.J., McCrimmon, D.R., 2006. Central pathways of pulmonary and lower airway vagal afferents. J Appl Physiol 101, 618 627. Lang, P.J., Bradley, M.M., Cuthbert, B.N., 2008. International affective picture system (IAPS): affective ratings of pictures and instruction manual. University of Florida, Gainesville, FL. Technical Report A 8 Lavietes, M.H., Sanchez, C.W., Tiersky, L.A., Cherniack, N.S., Natelson, B.H., 2000. Psychological profile and ventilatory response to inspiratory resistive loading. Am J Respir Crit Care Med 161, 737 744. Lipski, J., Kubin, L., Jodkowski, J., 1983. Synaptic action of R beta neurons on phrenic motone urons studied with spike triggered averaging. Brain Res 288, 105 118.

PAGE 124

124 Livermore, N., Butler, J.E., Sharpe, L., McBain, R.A., Gandevia, S.C., McKenzie, D.K., 2008. Panic attacks and perception of inspiratory resistive loads in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 178, 7 12. Lloyd, K.G., DeMontis, G., Broekkamp, C.L., Thuret, F., Worms, P., 1983. Neurochemical and neuropharmacological indications for the involvement of GABA and glycine receptors in neuropsychiatric disorders. Adv B iochem Psychopharmacol 37, 137 148. Lopata, M., Onal, E., Ginzburg, A.S., 1983. Respiratory muscle function during CO2 rebreathing with inspiratory flow resistive loading. J Appl Physiol 54, 475 482. Lynne Davies, P., Couture, J., Pengelly, L.D., West, D., Bromage, P.R., Milic Emili, J., 1971. Partitioning of immediate ventilatory stabiliy to added elactic loads in cats. J Appl Physiol 30, 814 819. Magadle, R., Berar Yanay, N., Weiner, P., 2002. The risk of hospitalization and near fatal and fatal asthma in relation to the perception of dyspnea. Chest 121, 329 333. Main, J., Moss Morris, R., Booth, R., Kaptein, A.A., Kolbe, J., 2003. The use of reliever medication in asthma: the role of negative mood and symptom reports. J Asthma 40, 357 365. Matsumoto, S., Takeda, M., Saiki, C., Takahashi, T., Ojima, K., 1997. Effects of vagal and carotid chemoreceptor afferents on the frequency and pattern of spontaneous augmented breaths in rabbits. Lung 175, 175 186. McCrimmon, D.R., Alheid, G.F., 2004. Capra, eupnea, dys pnea, apnea: respiratory rhythms and the pre Botzinger complex in the goat. J Appl Physiol 97, 1618 1619. McCrimmon, D.R., Speck, D.F., Feldman, J.L., 1987. Role of the ventrolateral region of the nucleus of the tractus solitarius in processing respiratory afferent input from vagus and superior laryngeal nerves. Exp Brain Res 67, 449 459. Merrill, E.G., Lipski, J., Kubin, L., Fedorko, L., 1983. Origin of the expiratory inhibition of nucleus tractus solitarius inspiratory neurones. Brain Res 263, 43 50. Molkov, Y.I., Bacak, B.J., Dick, T.E., Rybak, I.A., 2013. Control of breathing by interacting pontine and pulmonary feedback loops. Front Neural Circuits 7, 16. Ng, T.P., Niti, M., Tan, W.C., Cao, Z., Ong, K.C., Eng, P., 2007. Depressive symptoms and chron ic obstructive pulmonary disease: effect on mortality, hospital readmission, symptom burden, functional status, and quality of life. Arch Intern Med 167, 60 67.

PAGE 125

125 Nicoll, R.A., Malenka, R.C., Kauer, J.A., 1990. Functional comparison of neurotransmitter recep tor subtypes in mammalian central nervous system. Physiol Rev 70, 513 565. Otake, K., Nakamura, Y., Ezure, K., 1993. Projections from the commissural subnucleus of the solitary tract onto catecholamine cell groups of the ventrolateral medulla. Neurosci Let t 149, 213 216. Pate, K.M., Davenport, P.W., 2012a. Tracheal Occlusion Conditioning Causes Stress, Anxiety and Neural State Changes in Conscious Rats. Exp Physiol. Pate, K.M., Davenport, P.W., 2012b. Tracheal occlusions evoke respiratory load compensation and neural activation in anesthetized rats. J Appl Physiol 112, 435 442. Paton, J.F., Ramirez, J.M., Richter, D.W., 1994. Mechanisms of respiratory rhythm generation change profoundly during early life in mice and rats. Neurosci Lett 170, 167 170. Paton, J .F., Richter, D.W., 1995. Role of fast inhibitory synaptic mechanisms in respiratory rhythm generation in the maturing mouse. J Physiol 484 ( Pt 2), 505 521. Paxinos, G., Watson, C., 1998. The rat brain in stereotaxic coordinates. Phillipson, E.A., 1974. V agal control of breathing pattern independent of lung inflation in conscious dogs. J Appl Physiol 37, 183 189. Puddy, A., Giesbrecht, G., Sanii, R., Younes, M., 1992. Mechanism of detection of resistive loads in conscious humans. J Appl Physiol 72, 2267 22 70. Put, C., Van den Bergh, O., Van Ongeval, E., De Peuter, S., Demedts, M., Verleden, G., 2004. Negative affectivity and the influence of suggestion on asthma symptoms. J Psychosom Res 57, 249 255. Rekling, J.C., Feldman, J.L., 1998. PreBotzinger complex and pacemaker neurons: hypothesized site and kernel for respiratory rhythm generation. Annu Rev Physiol 60, 385 405. Richter, D.W., Ballanyi, K., Schwarzacher, S., 1992. Mechanisms of respiratory rhythm generation. Curr Opin Neurobiol 2, 788 793. Rietveld, S., Prins, P.J., 1998. The relationship between negative emotions and acute subjective and objective symptoms of childhood asthma. Psychol Med 28, 407 415.

PAGE 126

126 Rybak, I.A., Abdala, A.P., Markin, S.N., Paton, J.F., Smith, J.C., 2007. Spatial organization and s tate dependent mechanisms for respiratory rhythm and pattern generation. Prog Brain Res 165, 201 220. Rybak, I.A., O'Connor, R., Ross, A., Shevtsova, N.A., Nuding, S.C., Segers, L.S., Shannon, R., Dick, T.E., Dunin Barkowski, W.L., Orem, J.M., Solomon, I.C ., Morris, K.F., Lindsey, B.G., 2008. Reconfiguration of the pontomedullary respiratory network: a computational modeling study with coordinated in vivo experiments. J Neurophysiol 100, 1770 1799. Saether, K., Hilaire, G., Monteau, R., 1987. Dorsal and ven tral respiratory groups of neurons in the medulla of the rat. Brain Res 419, 87 96. Saetta, M., Turato, G., 2001. Airway pathology in asthma. Eur Respir J Suppl 34, 18s 23s. Saito, Y., Tanaka, I., Ezure, K., 2002. Morphology of the decrementing expiratory neurons in the brainstem of the rat. Neurosci Res 44, 141 153. Schreihofer, A.M., Stornetta, R.L., Guyenet, P.G., 1999. Evidence for glycinergic respiratory neurons: Botzinger neurons express mRNA for glycinergic transporter 2. J Comp Neurol 407, 583 597. Smith, B.K., Martin, A.D., Vandenborne, K., Darragh, B.D., Davenport, P.W., 2012. Chronic intrinsic transient tracheal occlusion elicits diaphragmatic muscle fiber remodeling in conscious rodents. PLoS One 7, e49264. Smith, J.C., Abdala, A.P., Koizumi, H., Rybak, I.A., Paton, J.F., 2007. Spatial and functional architecture of the mammalian brain stem respiratory network: a hierarchy of three oscillatory mechanisms. J Neurophysiol 98, 3370 3387. Smith, J.C., Butera, R.J., Koshiya, N., Del Negro, C., Wilson, C.G., Johnson, S.M., 2000. Respiratory rhythm generation in neonatal and adult mammals: the hybrid pacemaker network model. Respir Physiol 122, 131 147. Smith, J.C., Ellenberger, H.H., Ballanyi, K., Richter, D.W., Feldman, J.L., 1991. Pre Botzinger complex : a brainstem region that may generate respiratory rhythm in mammals. Science 254, 726 729. Smith, J.C., Abdala, A.P., Borgmann, A., Rybak, I.A., Paton, J.F., 2013. Brainstem respiratory networks: building blocks and microcircuits. Trends Neurosci 36, 152 162. St John, W.M., Stornetta, R.L., Guyenet, P.G., Paton, J.F., 2009. Location and properties of respiratory neurones with putative intrinsic bursting properties in the rat in situ. J Physiol 587, 3175 3188.

PAGE 127

127 Stornetta, R.L., 2008. Identification of neurot ransmitters and co localization of transmitters in brainstem respiratory neurons. Respir Physiol Neurobiol 164, 18 27. Subramanian, H.H., Chow, C.M., Balnave, R.J., 2007. Identification of different types of respiratory neurones in the dorsal brainstem nuc leus tractus solitarius of the rat. Brain Res 1141, 119 132. Sun, Q.J., Goodchild, A.K., Chalmers, J.P., Pilowsky, P.M., 1998. The pre Botzinger complex and phase spanning neurons in the adult rat. Brain Res 809, 204 213. Sykes, R.M., Spyer, K.M., Izzo, P. N., 1997. Demonstration of glutamate immunoreactivity in vagal sensory afferents in the nucleus tractus solitarius of the rat. Brain Res 762, 1 11. Takakura, A.C., Moreira, T.S., West, G.H., Gwilt, J.M., Colombari, E., Stornetta, R.L., Guyenet, P.G., 2007. GABAergic pump cells of solitary tract nucleus innervate retrotrapezoid nucleus chemoreceptors. J Neurophysiol 98, 374 381. Tanaka, I., Ezure, K., Kondo, M., 2003. Distribution of glycine transporter 2 mRNA containing neurons in relation to glutamic acid decarboxylase mRNA containing neurons in rat medulla. Neurosci Res 47, 139 151. Tian, G.F., Peever, J.H., Duffin, J., 1999. Botzinger complex, bulbospinal expiratory neurones monosynaptically inhibit ventral group respiratory neurones in the decerebrate ra t. Exp Brain Res 124, 173 180. Tiller, J., Pain, M., Biddle, N., 1987. Anxiety disorder and perception of inspiratory resistive loads. Chest 91, 547 551. Turato, G., Zuin, R., Saetta, M., 2001. Pathogenesis and pathology of COPD. Respiration 68, 117 128. v an der Kooy, D., Koda, L.Y., McGinty, J.F., Gerfen, C.R., Bloom, F.E., 1984. The organization of projections from the cortex, amygdala, and hypothalamus to the nucleus of the solitary tract in rat. J Comp Neurol 224, 1 24. Van Diest, I., Janssens, T., Bogaerts, K., Fannes, S., Davenport, P.W., Van Den Bergh, O., 2009. Affective modulation of inspiratory motor drive. Psychophysiology 46, 12 16. Vogele, C., von Leupoldt, A., 2008. Mental disorders in chronic obstructive pulmonary disease (COPD). Respir Me d 102, 764 773. von Leupoldt, A., Chan, P.Y., Bradley, M.M., Lang, P.J., Davenport, P.W., 2011. The impact of anxiety on the neural processing of respiratory sensations. Neuroimage 55, 247 252.

PAGE 128

128 von Leupoldt, A., Dahme, B., 2005. Cortical substrates for the perception of dyspnea. Chest 128, 345 354. von Leupoldt, A., Dahme, B., 2007. Psychological aspects in the perception of dyspnea in obstructive pulmonary diseases. Respir Med 101, 411 422. von Leupoldt, A., Mertz, C., Kegat, S., Burmester, S., Dahme, B., 2006a. The impact of emotions on the sensory and affective dimension of perceived dyspnea. Psychophysiology 43, 382 386. von Leupoldt, A., Riedel, F., Dahme, B., 2006b. The impact of emotions on the perception of dyspnea in pediatric asthma. Psychophysiolo gy 43, 641 644. von Leupoldt, A., Sommer, T., Kegat, S., Baumann, H.J., Klose, H., Dahme, B., Buchel, C., 2008. The unpleasantness of perceived dyspnea is processed in the anterior insula and amygdala. Am J Respir Crit Care Med 177, 1026 1032. von Leupoldt A., Taube, K., Henkhus, M., Dahme, B., Magnussen, H., 2010. The impact of affective states on the perception of dyspnea in patients with chronic obstructive pulmonary disease. Biol Psychol 84, 129 134. Wagena, E.J., Arrindell, W.A., Wouters, E.F., van Sc hayck, C.P., 2005. Are patients with COPD psychologically distressed? Eur Respir J 26, 242 248. Watts, T.L., Wozniak, J.A., Davenport, P.W., Hutchison, A.A., 1997. Laryngeal and diaphragmatic activities with a single expiratory load in newborn lambs. Respi r Physiol 107, 27 35. Webb, B., Hutchison, A.A., Davenport, P.W., 1994. Vagally mediated volume dependent modulation of inspiratory duration in the neonatal lamb. J Appl Physiol 76, 397 402. Webb, B., Hutchison, A.A., Davenport, P.W., 1996. Contribution of vagal afferents to the volume timing response to expiratory loads in neonatal lambs. Neurosci Lett 207, 147 150. Webster, K.E., Colrain, I.M., 2000. The relationship between respiratory related evoked potentials and the perception of inspiratory resistive loads. Psychophysiology 37, 831 841. Yu, J., 2005. Airway mechanosensors. Respir Physiol Neurobiol 148, 217 243. Zechman, F.W., Frazier, D.T., Lally, D.A., 1976. Respiratory volume time relationships during resistive loading in the cat. J Appl Physiol 40, 177 183.

PAGE 129

129 BIOGRAPHICAL SKETCH Hsiu Wen Tsai was born in Hsinc h u Taiwan. She received her Bachelor of Science degree in n utrition and h uman s ciences in Taipei Medical University in T aipei Taiwan in 2002 She received her Master of Science degree in b iochemistry and m olecular b iology 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 res piratory physiology and neurophysiology since August 200 8 Her research interests include neurotransmission, respiratory sensation and respiratory mechanism.